Tumor vaccination in combination with hematopoietic cell transplantation for cancer therapy

ABSTRACT

In one aspect, the present invention provides a method for treating cancer comprising tumor cell vaccination in combination with hematopoietic and immune cell transplantation. In some embodiments, the method involves autologous tumor cell vaccination prior to autologous hematopoietic and immune cell transplantation. In another aspect, the present invention provides a method of purifying tumor cells from a subject in preparation for vaccination.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract CA049605awarded by the National Institutes of Health. The Government has certainrights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 27, 2010, is named13279712.txt, and is 551 bytes in size.

BACKGROUND OF THE INVENTION

Cancer, also known as malignant neoplasm, is characterized by anabnormal growth of cells that display uncontrolled cell division,invasion and destruction of adjacent tissues, and sometimes metastasisto other locations in the body. There are more than 100 types of cancer,including breast cancer, skin cancer, lung cancer, colon cancer,prostate cancer, and lymphoma. Cancer is the second leading cause ofdeath in America and it causes about 13% of all deaths. Cancer mayaffect people at all ages, even fetuses, but the risk for most types ofcancer increases with age. Cancers can affect all animals.

Chemotherapy has become the standard of care for many cancers.Chemotherapy refers to antineoplastic drugs used to treat cancer or thecombination of these drugs into a cytotoxic standardized treatmentregimen. Most commonly, chemotherapy acts by killing cells that dividerapidly, one of the main properties of cancer cells. This means that italso harms cells that divide rapidly under normal circumstances: cellsin the bone marrow, digestive tract and hair follicles; this results inthe most common side effects of chemotherapy—myelosuppression (decreasedproduction of blood cells), mucositis (inflammation of the lining of thedigestive tract) and alopecia (hair loss). Newer anticancer drugs actdirectly against abnormal proteins in cancer cells; this is termedtargeted therapy.

Despite these new agents and improved combinations, the currenttreatment is still not effective for many types of cancers or cancers atdifferent stages. Improved regimens and treatments are greatly neededfor cancer therapy.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for treatingcancer comprising: (a) obtaining purified tumor cells; (b) vaccinating asubject with cancer with their purified tumor cells that have beenirradiated and combined with an adjuvant; (c) collecting immune andhematopoietic cells from the vaccinated subjects; and (d) injecting thecollected immune and hematopoietic cells from the subject intravenouslyafter total body irradiation of the recipient. In some embodiments, thetumor cells are purified from a tumor tissue in a tumor-bearing subject.In some embodiments, the tumor cells are purified away from stromalcells. In some embodiments, the tumor cells are purified away fromimmunosuppressive cells. In some embodiments, the purified tumor cellsare irradiated and stimulated prior to vaccination. In some embodiments,the purified tumor cells are combined with an adjuvant. The adjuvant canbe CpG or GM-CSF or other immunostimulants. In some embodiments, thedonor subject is tumor-bearing. In some embodiments, the immune cellscontain T cells. In some embodiments, the immune cells are added tohematopoietic progenitor cells, for example, CD34⁺ cells. In someembodiments, the hematopoietic cells are mobilized in the vaccinatedsubject and enriched prior to transplantation. In some embodiments, therecipient receives a single or several doses of total body irradiationprior to transplantation with or without local irradiation of the tumor.In some embodiments, the subject is a patient diagnosed with cancer. Ina preferred embodiment, the transplantation is an autologoustransplantation of T cells and hematopoietic progenitor cells. In someembodiments, the subject method further comprises vaccinating thesubject with irradiated tumor cells and adjuvant before thetransplantation of immune and hematopoietic cells. In some embodiments,the cancer is a solid tumor. Examples of solid tumors that can betreating using the subject methods of the present invention include butare not limited to colorectal cancer, lung cancer, breast cancer,pancreatic cancer, liver cancer, prostate cancer, and ovarian cancer. Insome embodiments, the tumor cells are from a primary or metastatictumor. In some embodiments, the cancer is primary or metastatic.

In another aspect, the present invention provides a method for purifyingtumor cells from vaccination comprising: (a) obtaining a tumor tissuefrom a subject; (b) making cell suspension of the tumor tissue; (c)separating tumor cells from the cell suspension; and (d) obtainingpurified tumor cells with a purity of at least 30%. In some embodiments,the tumor is a primary tumor or a metastatic tumor. In some embodiments,the tumor is a solid tumor. The solid tumor can be a colorectal tumor,breast tumor, lung tumor, liver tumor, pancreatic tumor, prostate tumor,or ovarian tumor. In some embodiments, the subject is a patientdiagnosed with cancer. In some embodiments, the tumor cells areseparated from other components in the cell suspension. In someembodiments, the tumor cells are purified from immunosuppressive cellsand factors present in the cell suspension. In some embodiments, thetumor cell purity is greater than 30%, 40%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the tumor cellpurity is greater than 90%. In some embodiments, the purified tumorcells are subsequently used for vaccinating the subject from whom thetumor tissue is originally obtained. In some embodiments, the purifiedtumor cells are irradiated and stimulated with an adjuvant. The adjuvantcan be CpG or GM-CSF or other immunostimulants.

In another aspect, the present invention provides a compositioncomprising purified and irradiated tumor cells from a subject. In someembodiments, the tumor cells are purified from stromal cells. In someembodiments, the tumor cells are purified from immunosuppressive cells.In some embodiments, the purified and irradiated tumor cells arestimulated prior to vaccination. In some embodiments, the purified tumorcells are combined with an adjuvant. The adjuvant can be CpG or GM-CSFor other immunostimulant. In some embodiments, the subject is a patientdiagnosed with cancer. In some embodiments, the purified and irradiatedtumor cells are used to vaccinate the subject. In some embodiments, thepurified and irradiated tumor cells are from a solid tumor. The solidtumor includes but is not limited to colorectal tumor, lung tumor,breast tumor, pancreatic tumor, liver tumor, prostate tumor, and ovariantumor. In some embodiments, the purified and irradiated tumor cells arefrom a primary or metastatic tumor. In some embodiments, the purifiedtumor cells have a purity greater than 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95%.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the United States Patent andTrademark Office upon request and payment of the necessary fee.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A shows the experimental scheme, which uses HCT fromtumor-vaccinated donors to treat CT26 colon tumors in syngeneic mice. Inall instances, normal BALB/c donor mice were vaccinated subcutaneously(s.c.) with 10⁶ irradiated CT26 tumor cells mixed with 30 μg CpG, anadjuvant that stimulates antigen presenting cell via TLR-9 (12,13).After 90 days, spleen and bone marrow cells were harvested, andtransplanted intravenously (i.v.) into tumor-bearing BALB/c host micefollowing a single dose of total body irradiation (TBI). Seven daysprior to TBI, hosts had been given live tumor cells via s.c. (2.5×10⁴),i.v. (2×10⁵) or intrasplenic (5×10⁵) routes. FIG. 1B shows theprogressive growth of s.c. tumors in all untreated mice. Similarly,tumor bearing recipients of 50×10⁶ bone marrow cells and 60×10⁶ spleencells from unvaccinated donors had uniformly progressive tumors (FIG.1C). In contrast, after HCT from vaccinated donors, tumor bearing micedisplayed a steady regression of tumor volume over a 100 day observationperiod (FIG. 1D), which remained stable until the end of study (day 180;data not shown). Shortening the time interval between immunization ofthe donor and harvesting the graft from 90 to 14 days, but not to 50days, resulted in lower anti-tumor effect (p=0.005 and p=0.3,respectively; log rank test; FIG. 1E). Omission of CpG from the donorvaccine resulted in a further loss of efficacy (p=0.01), and only 20% ofhosts survived 100 days.

The same HCT strategy was also successful in recipients given tumorcells by i.v. administration. By day 7, tumor cells had disseminatedinto the lungs and formed multiple tumor clusters (FIG. 1H). By day 20all untreated control mice succumbed to progressive disease with large,nearly confluent tumor nodules (FIGS. 1F and 1H). In contrast,recipients of HCT from vaccinated donors all survived at least 100 days,with no histologic evidence of residual tumor (FIG. 1H). Accordingly,improvement of survival was significant as compared to untreated mice(p<0.01) (FIG. 1F). When tumor cells were injected into the spleen, byday 7 tumor nodules became established in the parenchyma of the liver(FIG. 1I), and by day 14 there was evidence of blood vessel invasion(arrows, FIG. 1I). All untreated animals died by day 30 (FIG. 1G) withmultiple visible, as well as microscopic, tumors. Treated mice survivedbeyond day 100 (FIG. 1G), easily exceeding the survival of untreatedmice (p=0.001). The liver of treated mice displayed no abnormalities andalso no histologic evidence of residual tumor at day 60 (FIG. 1I). HCTfrom vaccinated donors also cured peritoneal carcinomatosis, which hadbeen created by intraperitoneal injection of 5×10⁶ tumor cells and whichdisplayed multiple peritoneal nodules and ascites by the time oftransplant (data not shown). All untreated mice died by day 20, and alltransplanted mice survived at least 100 days without any peritonealtumor growth.

FIG. 2 shows that “cured” animals are protected from tumor challenge andcan serve as donors of immune cells for HCT into syngeneic tumor-bearinghosts. FIG. 2A shows the animals with a single subcutaneous nodulescured by HCT from vaccinated animals were challenged at day 120 withlive tumor cells subcutaneously. p<0.001 for “cured” animals (n=5) vsuntreated controls (n=9). FIG. 2B shows the bone marrow and splenocytesfrom “cured” animals were harvested at day 180 and transferred intolethally irradiated tumor-bearing hosts. 100 days after bone marrow andsplenocytes were harvested from secondary hosts and transferred intolethally irradiated tumor-bearing hosts. p<0.001 for transplantedanimals (n=5) vs untreated (n=9) controls.

To assess the duration of effect of vaccination combined with HCT,“cured” animals from the experiment in FIG. 1D were challenged with2.5×10⁴ live tumor cells at day 120 as shown in the experimental schemein FIG. 2A. The results show that these animals were completelyprotected and survived for at least 100 days (FIG. 2A). Moreover,harvesting of spleen and bone marrow cells from “cured” recipients atday 180 after HCT, and secondary transfer resulted in 80% of the newrecipients surviving for more than 100 days (FIG. 2B). At least 100 dayslater those secondary recipients were used as donors for another HCTinto irradiated tumor-bearing tertiary hosts which was also effectivesince all tertiary hosts survived more than 100 days (FIG. 2B). Thus,anti-tumor immunity generated by a single vaccination could eradicatetumors 370 days later (FIG. 2B).

FIG. 3 shows requirements for host irradiation and donor T cells intransplants. FIG. 3A shows the survival of BALB/c hosts that receivedeither 800 cGy TBI (n=5), 450 cGy TBI (n=5) or no conditioning (n=5)before transplantation. p<0.05 for 800 cGy vs 450 cGy and p<0.0001 for800 cGy vs 0 cGy. FIG. 3B shows the Rag 2^(−/−) BALB/c hosts receivedtransplants after either 800 cGy TBI (n=5) or no irradiation (n=5).p=0.001 for irradiated RAG2^(−/−) vs unirradiated. FIG. 3C shows theBALB/c hosts received transplants from BALB/c IFN-γ^(−/−) donors (n=10)(p=0.004), DBA/2J donors (n=5) (p<0.001) or BALB/c donors vaccinatedwith either AH-1 peptide and CpG (n=6) (p<0.0001) or irradiated tumorcells and lipopolysaccharide (LPS) instead of CpG (n=5) (p=1.0).(Survival compared with 90 day CpG group from FIG. 1E). FIG. 3D showsthe hosts were given either donor whole bone marrow cells (n=5), orc-kit⁺Sca-1⁺ HSCs alone (n=5), or HSCs and purified splenic T cells(n=6) (p=0.008). FIG. 3E shows the hosts received HSCs with either CD4⁺(n=10) or CD8⁺ (n=8) T cells, or transplants containing a mixture ofCD4⁺ and CD8⁺ T cells (n=5) (p<0.008). FIG. 3F shows the survival ofC57BL/6 mice with MC38 subcutaneous colon tumor nodules given TBI andtransplants from C57BL/6 donors that had been vaccinated with irradiatedMC38 tumor cells and CpG (n=10) as compared with untreated control mice(n=5). (p<0.008). FIG. 3G shows the survival of hosts (n=5) with largesubcutaneous tumors established for 15 days after HCT from vaccinateddonors compared with survival of untreated animals (p=0.0005).

FIG. 3A illustrates the significant role of the host conditioningregimen in our vaccine strategy. All tumor bearing recipients of bonemarrow and splenocytes from vaccinated donors were cured whenconditioned with myeloablative TBI (800 cGy). In contrast, only 60% ofhosts survived 100 days with a non-myeloablative radiation dose (450cGy) (p<0.05), while none survived more than 40 days without irradiation(p<0.0001). Radiation causes lymphodepletion which might deplete hostregulatory T cells that suppress anti-tumor immune responses. In orderto test whether radiation mediates tumor eradication through such amechanism, we studied unirradiated tumor-bearing RAG2^(−/−) recipientsthat lack T cells. Rag 2^(−/−) BALB/c hosts were given myeloablativeradiation or no radiation immediately before transplantation of cellsfrom vaccinated donors. FIG. 3B shows that although there was asignificant delay in mortality in non-irradiated Rag 2−/− mice ascompared to non-irradiated wild type mice (p=0.001), all mice died byday 65. Conditioning of the Rag 2^(−/−) mice with 800 cGy resulted insignificant improvement in survival as compared to the non-irradiatedmice (p=0.001), and all hosts survived at least 100 days (FIG. 3B).

When IFNγ^(−/−) BALB/c mice were vaccinated and used as bone marrow andsplenocyte donors, the survival of hosts was decreased as compared towild type syngeneic donors (p=0.004) (FIG. 2C). Likewise, survival wasreduced in all five mice given grafts from vaccinated MHC-matched, minorantigen-mismatched DBAJ2J donors (H-2^(d)) (p<0.001) (FIG. 3C). While4/5 animals had progressive tumor growth, one mouse displayed tumorregression, but succumbed due to GVHD. Substitution of whole tumor cellswith the tumor-associated immunodominant AH-1 peptide (4) forvaccination of donors resulted in decreased survival (p<0.0001) (FIG.3C). Use of lipopolysaccharide (30 μg) as an adjuvant for vaccinationwas just as effective as CpG, based on survival of hosts post-HCT(p=1.0) (FIG. 3C). Grafts from vaccinated donors consisting of bonemarrow or FACS-purified c-kit⁺Sca1^(high)lin⁻hematopoietic stem cells(HSC) failed to prevent tumor progression (FIG. 3D). Addition of 5×10⁶splenic T cells to the HSCs increased survival (p=0.008) such that themajority of hosts were alive at 100 days without detectable tumors (FIG.3D). When HSC grafts were supplemented with both CD4⁺ (3.5×10⁶) plusCD8⁺ T cells (1.8×10⁶), survival was improved (p<0.008) as compared tosupplementation with CD4⁺ or CD8⁺ T cells only (FIG. 3E).

The utility of HCT from vaccinated donors was further validated instudies of another colon cancer, MC38, which grows only in C57BL/6(H-2^(b)) mice (8). For these experiments, donor mice were vaccinatedwith 1×10⁶ MC38 tumor cells (FIG. 3F). Again, while syngeneic recipientsof grafts from vaccinated donors were cured, recipients of transplantsfrom unvaccinated donors did not survive beyond 35 days (p<0.0001). HCTfrom tumor-vaccinated donors could also significantly improve survivalof animals with large (>10 mm) tumors established for 15 days (p=0.0005)(FIG. 3G). After 100 days, 60% of treated animals were completely tumorfree.

FIG. 4 shows that donor T cells accumulate in host tumors aftertransplantation. FIG. 4A shows the experimental scheme. FIG. 4B showsthe Thy 1.1 and Thy 2.2 analysis of single cell suspensions of thetumors and spleens obtained on day 28 from tumor-bearing hosts givenunvaccinated or vaccinated donor transplants with or without TBI. FIG.4C shows the mean percentages and SE of tumor-infiltrating Thy1.1⁺ cellsat day 28 in top panel (n=5 in each group). Mean absolute numbers ofThy1.1⁺ cells in the host spleen in bottom panel (n=5). (p<0.001 fordifferences between TBI and no TBI, and p>0.05 for unvaccinated donorsvs vaccinated after TBI). FIGS. 5D and 5E show the analysis of CD4⁺ andCD8⁺ T cells among Thy1.1⁺ cells. Vaccinated donors are compared withunvaccinated donors (n=5 in each group) (p<0.001 for tumors and p<0.001for spleens).

To delineate the donor-derived cell populations involved in theanti-tumor response, bone marrow and spleen cells were transplanted fromBALB/c Thy1.1 donors into tumor bearing BALB/c Thy 1.2 hosts, asdepicted in FIG. 4A. To assure that there would be sufficient donorcells for analysis at day 28, HCT was performed in animals bearingtumors that had been established for 14 instead of 7 days. In irradiatedrecipients of vaccinated donor grafts approximately 70% of T cellsinfiltrating the tumors were of donor origin (FIGS. 4B and 4C), whiledonor T cells accounted for <2% of tumoral T cells when hosts were notirradiated (p<0.001). A similar facilitation of donor cell accumulationin the spleen was observed in irradiated versus non-irradiated hosts(p<0.01) (FIGS. 4B and 4C). Differences in accumulation of total T cellsfrom vaccinated and unvaccinated donors were not significant (p>0.05).However, the majority of tumor T cells from vaccinated donors were CD8⁺,whereas most of the tumoral T cells from unvaccinated donors were CD4⁺(p=0.01) (FIGS. 4D and 4E). CD4⁺ T cells were in the majority in thespleens with both vaccinated and unvaccinated donors (p<0.001).

FIG. 5 shows that the effector/regulatory T cell ratios changes in favorof CD8 effector memory cells as a result of vaccination and HCT. FIG. 5Ashows the experimental scheme. Syngeneic Thy 1.1 donors were vaccinatedwith 10⁶ irradiated CT26 cells and CpG. Bone marrow and splenocytes weretransplanted into lethally irradiated Thy 1.2 hosts with s.c. tumorsestablished for 14 days. Tumor-infiltrating cells and host spleens wereanalyzed d 14 after HCT (28 days after tumors were induced). Controltumor-bearing animals did not receive irradiation and HCT. FIG. 5B showsthe analysis of CD62L expression on CD8⁺ and CD4⁺ tumor-infiltrating Tcells form irradiated hosts that received HCT (gated Thy1.1⁺ cells) oruntreated control animals (no HCT) (gated Thy1.2⁺ cells) at day 28. Thedata are representative for the group of animals (n=5). FIGS. 5C and 5Dshow the CD25 expression of tumor-infiltration CD4+ T cells and analysisof CD4⁺CD25⁺Foxp3⁺ T cells in tumors obtained from untreated animals onday 14, untreated animals on day 28 as well as tumor-bearing animalsthat received HCT (day 28). Mean percentages and SE are shown (n=5 ineach group). FIG. 5E shows the CD8⁺ effector memory/Treg ratio in tumorsin animals receiving HCT versus untreated animals on day 28. Meanpercentages and SE are shown (n=5 in each group) (p<0.05).

Previous studies have shown that CD4⁺CD25⁺FoxP3⁺ Treg cells can suppresstumor immunity (14). Moreover, this suppression was mediated at thetumor site and was lost after intra-tumoral depletion of Tregs (14). Itis shown above (FIG. 3B) that conditioning and HCT was required to curetumors in RAG2^(−/−) mice lacking T regulatory cells. Thus, therequirement for irradiation is not based on host Treg depletion.

However, regulatory T cells of donor or host origin may be capable ofinfiltrating tumors when wild-type hosts are used. Host and donor T cellsubsets infiltrating CT26 subcutaneous tumor nodules were examined inwild-type BALB/c mice before and after HCT, and in controls without HCTas shown in the experimental scheme in FIG. 5A. Control Thy1.2 micegiven CT26 cells subcutaneously were euthanized 14 or 28 days later, andsingle cell suspensions from tumors were analyzed for tumor infiltratingT lymphocytes (TIL) subsets.

Experimental mice were lethally irradiated and given HCT from vaccinatedThy 1.1 donors after 14 days of tumor growth, and tumor cell suspensionwere analyzed 14 days after HCT. FIG. 5B shows the representativestaining patterns for CD4⁺ and CD8⁺ T cells in cell suspensions usinggated Thy1.2⁺ T cells from control mice and gated Thy1.1⁺ from micegiven HCT at 28 days after the subcutaneous injection of tumor cells (14days after HCT).

Whereas CD8⁺ and CD4⁺ cells accounted for about 90% and 5% respectivelyof Thy1.1⁺ cells in mice given HCT, the CD8⁺ and CD4⁺ cells accountedfor 30% and 28% respectively of Thy1.2⁺ cells in mice without HCT.Almost all of the CD8⁺ and CD4⁺ T cells in mice given HCT wereCD62L^(lo) (FIG. 5B). The staining pattern indicates that few nave orcentral memory cells were found in these tumors, almost all wereeffector memory cells, since the CD8⁺ and CD4⁺ cells were almost allCD44^(hi).

In contrast, the gated CD8⁺ and CD4⁺ cells from tumors in control micecontained discrete subsets of both CD62 L^(low) and CD62 L^(hi) cells.The CD62 L^(hi) cells accounted for 26% of CD8⁺ cells and 58% of CD4⁺cells (FIG. 5B). Staining of gated CD4⁺ tumor cells from control miceand those given HCT for CD4 versus CD25 showed that about 16% of CD4⁺cells were CD25⁺ in controls, and 32% were CD25⁺ in those given HCT atthe day 28 time point (FIG. 5C). At day 14, 22% of CD4⁺ cells wereCD25⁺. The results of additional staining for intracellular FoxP3⁺showed that the mean percentage of CD4⁺CD25⁺ FoxP3⁺ Treg cells amonggated CD4+ cells in the tumors of all 3 groups of mice varied from about15% to 25% (FIG. 5D).

The differences in the means were not statistically significant (p>0.05)as judged by the Student t test. Despite the similar percentages of Tregcells among total CD4+ T cells in the tumor cell infiltrate, there was amarked difference in the balance of CD8⁺CD62 L^(lo)CD44^(hi) effectormemory T cells versus Treg cells. Whereas, day 28 tumors from controlmice showed a mean ratio of about 5:1 CD8⁺ effector memory to Treg cellsin the infiltrate, the day 28 tumors from mice given HCT showed meanratio of about 50:1. The differences in ratios were statisticallysignificant (p<0.05). Thus, while HCT did not deplete Tregs at the tumorsite, the balance of tumor infiltrating cells was altered to favor CD8⁺effector memory T cells as compared to Treg cells.

FIG. 6 shows that tumor vaccination without HCT is not effective, butvaccination combined with HCT is highly effective, even in the presenceof growing tumor. FIG. 6A shows the experimental scheme. Tumors wereinduced subcutaneously at day 0. Animals were unvaccinated or vaccinatedat day 7 with irradiated tumor cells and CpG. FIG. 6B shows the survivalof vaccinated versus unvaccinated mice (n=8). FIG. 6C shows the nontumor-bearing animals were vaccinated with irradiated tumor cells andCpG, and challenged with live tumor cells 16 (n=5), 50 (n=5) or 90(n=10)days after vaccination. p=0.03 for 90 days vaccination vs control. FIG.6D shows the survival of vaccinated mice after tumor challenge. FIG. 6Eshows the experimental scheme. Mice with subcutaneous tumors werevaccinated at day 7 after tumor was induced. Tumors were resected at day21. At day 110 after tumor induction, bone marrow and splenocytes wereharvested and transferred into lethally irradiated tumor-bearing hosts,FIG. 6F shows the survival of hosts given bone marrow and spleen celltransplants from vaccinated or unvaccinated donors (n=5 each group)(p<0.05). FIG. 6G shows the donors were injected with tumor cells on day0, vaccinated on day 7 and splenectomized on day 14. Splenectomizeddonors had their abdominal incisions closed with surgical sutures beforereceiving a single dose of 800 cGy TBI. Within 6 hours of TBI, thedonors were given an autotransplant of all harvested spleen cellsinjected intravenously. FIG. 6H shows the survival of hosts with (n=7)and without (n=8) autologous HCT (p<0.001).

FIG. 6A shows the experimental scheme used to determine the effect ofvaccination alone on survival of tumor-bearing mice. FIG. 6B shows thatthe survival of vaccinated, but not HCT treated, animals with 7-daytumors did not improve as compared to unvaccinated tumor-bearing animalsand all animals died by day 40. Moreover, when vaccinated nontumor-bearing animals were challenged with as few as 2.5×10⁴ CT26 cells16 and 50 days after vaccination (FIG. 6C), only 20% of mice survived100 days (FIG. 6D). Some degree of protection developed in animalsvaccinated given the tumor challenge 90 days after vaccination, asindicated by the observation that 50% of animals remained tumor free(p=0.03) (FIG. 6D). Next, to assess the potential effect of largertumors on the response to vaccination, we vaccinated mice with tumorsgrowing for 7 days and then waited 14 additional days before resectingthe growing tumors at day 21 (FIG. 6E). Bone marrow and splenocytes wereharvested from donors on day 110 and transferred into lethallyirradiated tumor-bearing hosts. All hosts survived with complete tumorregression for at least 100 days (FIG. 6F). Only 20% of hosts giventransplants from unvaccinated donors survived 100 days, and thedifference using vaccinated versus unvaccinated donors was significant(p<0.05) (FIG. 6F). Thus, HCT from vaccinated animals into syngeneictumor bearing hosts resulted in cure of tumors, indicating that growingtumors in donors do not prevent the development of potent anti-tumoreffector cells in adoptive hosts.

A model of autolous HCT was studied as shown in FIG. 6G. In this schemea group of donors was vaccinated 7 days after live tumor cell injection,splenectomized 7 days later, conditioned with TBI immediately afterrecovery from surgery, and spleen cells were injected intravenouslywithin 6 hours after TBI. Bone marrow cells were not required for rescueof these myeloablated hosts, since mouse spleen cells contain bothimmune cells and HSCs. Donors that received the autologous transplantshad significantly improved survival as compared to those withouttransplants, and about 40% survived at least 100 days with completetumor regression (p<0.001) (FIG. 6H). Thus, large 14 day tumors wereeither cured or their growth significantly delayed after autologous HCT.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for treating cancer comprisingthe steps of a) obtaining purified tumor cells; b) vaccinating a donorsubject with the purified tumor cells; c) collecting immune cells fromthe vaccinated donor; and d) transplanting the collected hematopoieticcells and immune cells from the donor into a tumor-bearing recipientfollowing total body irradiation of the recipient. Also provided by thepresent invention is a method for purifying tumor cells from vaccinationcomprising: a) obtaining a tumor tissue from a subject; b) making cellsuspension of the tumor tissue; and c) separating tumor cells from thecell suspension; and d) obtaining purified tumor cells with a purity ofat least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or more.

In some embodiments, the tumor cells are purified from a tumor tissue ina tumor-bearing subject. In some embodiments, the tumor cells arepurified by density gradients using Ficoll or Percoll—followed bycentrifugation. In some embodiments, the tumor cells are purified bystaining for cell surface markers that recognize tumor cells, andsubsequent separation of positive staining cells. Followingpurification, tumor cells are irradiated and then stimulated with anadjuvant. Examples of adjuvant that can be used in the subject methodsof the present invention include but are not limited to varioustoll-like receptor (TLR) stimulants such as CpG, Lipopolysaccharide(LPS), poly-IC, and cytokines such as granulocyte-macrophagecolony-stimulating factor (GM-CSF).

In some embodiments, the present invention provides a method of treatingcancer comprising obtaining purified tumor cells from a cancer patient;vaccinating the patient with the tumor cells mixed with an adjuvant;collecting hematopoietic cells and immune cells from the vaccinatedpatient; and performing autologous transplantation of the collectedhematopoietic cells and immune cells back into the patient followingtotal body irradiation of the patient. In some embodiments, the immunecells are T cells. In some embodiments, the hematopoietic cells areCD34⁺ progenitor cells. Examples of cancer that can be treated by thesubject methods of the present invention include but are not limited tosolid tumors such as colorectal cancer, lung cancer, pancreatic cancer,breast cancer, prostate cancer, liver cancer, and ovarian cancer. Thetumor can be primary or metastatic.

Cancer Immunotherapy

In one aspect, the present invention discloses a method for treatingcancer via tumor vaccination followed by autologous hematopoietic andimmune cell transplantation. The method comprises a) obtaining purifiedtumor cells; b) vaccinating a donor subject with the purified tumorcells; c) collecting immune cells from the vaccinated donor; and d)transplanting the collected immune cells from the donor into atumor-bearing recipient following total body irradiation of therecipient.

Cancer immunotherapy is the use of the immune system to reject cancer.The main premise is stimulating the patient's immune system to attackthe malignant tumor cells that are responsible for the disease. This canbe either through immunization of the patient, in which case thepatient's own immune system is trained to recognize tumor cells astargets to be destroyed, or through the administration of therapeuticantibodies as drugs, in which case the patient's immune system isrecruited to destroy tumor cells by the therapeutic antibodies.

Since the immune system responds to the environmental factors itencounters on the basis of discrimination between self and non-self,many kinds of tumor cells that arise as a result of the onset of cancerare more or less tolerated by the patient's own immune system since thetumor cells are essentially the patient's own cells that are growing,dividing and spreading without proper regulatory control. In spite ofthis fact, however, many kinds of tumor cells display unusual antigensthat are either inappropriate for the cell type and/or its environment,or are only normally present during the organisms' development (e.g.fetal antigens). Examples of such antigens include but are not limitedto the glycosphingolipid GD2, a disialoganglioside that is normally onlyexpressed at a significant level on the outer surface membranes ofneuronal cells, where its exposure to the immune system is limited bythe blood-brain barrier. GD2 is expressed on the surfaces of a widerange of tumor cells including neuroblastoma, medulloblastomas,astrocytomas, melanomas, small-cell lung cancer, osteosarcomas and othersoft tissue sarcomas. Other kinds of tumor cells display cell surfacereceptors that are rare or absent on the surfaces of healthy cells, andwhich are responsible for activating cellular signal transductionpathways that cause the unregulated growth and division of the tumorcell. Examples include ErbB2, a constitutively active cell surfacereceptor that is produced at abnormally high levels on the surface ofbreast cancer tumor cells.

Vaccines have been tested to be able to induce integrated immuneresponses composed of target-specific antibodies and CD4+ and CD8+ Tlymphocytes, all of which are held to be essential for effectivelong-term control of cancer. Insights from these studies have generateda strong framework for the selection of components that will likelycomprise an ideal therapeutic cancer vaccine, including: multiplecancer-antigens in various forms delivered with potent adjuvants and alladministered in a prime-boost setting in conjunction with a modulator ofcancer immunosuppression. In one example, a skin cancer patient has beentreated using immune cells cloned from his own immune system, i.e.autologous cells, which were then re-injected into the patient. The term“autologous” in the context of transplantation typically refers to thesituation in which the donor and recipient are the same person. Anautologous graft is providing a graft, for example of skin, to the sameperson from which the graft is obtained. The patient, who was sufferingfrom advanced skin cancer, was free from tumors within eight weeks ofbeing injected with autologous immune cells. This result implicates thatautologous transplantation can be an effective treatment of cancer ingeneral.

Another approach to therapeutic anti-cancer vaccination is to generatethe immune response in situ in the patient. One example is OncoVEXGM-CSF. OncoVEX GM-CSF is a version of herpes simplex virus which hasbeen engineered to replicate selectively in tumor tissue and also toexpress the immune stimulatory protein GM-CSF. This enhances theanti-tumor immune response to tumor antigens released following lyticvirus replication providing an in situ, patient specific anti-tumorvaccine as a result. Effective cancer vaccines seek to target an antigenspecific to the tumor and distinct from self-proteins. Selection of theappropriate adjuvant, molecules that activate antigen-presenting cellsto stimulate immune responses, is required; at the present time, onlyBCG, aluminum-based salts and a squalene-oil-water emulsion are approvedworldwide for clinical use. The effective vaccine also should seek toprovide long term memory to prevent tumor recurrence. Preferably, boththe innate and adaptive immune systems should be activated(Pejawar-Gaddy S, Finn O. (2008) Critical Reviews in OncologyHematology. 67: 93-102).

Tumor antigens have been divided into two broad categories: shared tumorantigens; and unique tumor antigens. Shared antigens are expressed bymany tumors. Unique tumor antigens result from mutations induced throughphysical or chemical carcinogens; they are therefore expressed only byindividual tumors.

In one approach, vaccines contain whole tumor cells, though thesevaccines have been less effective in eliciting immune responses inspontaneous cancer models. Defined tumor antigens decrease the risk ofautoimmunity but because the immune response is directed to a singleepitope, tumors can evade destruction through antigen loss variance. Aprocess called “epitope spreading” or “provoked immunity” may mitigatethis weakness, as sometimes an immune response to a single antigen willlead to development of immunity against other antigens on the sametumor. Most of the cancer vaccines in development are addressingspecific cancer types and are therapeutic vaccines. Several cancervaccines are currently in development by companies such as AntigenicsInc. (Oncophage), Geron Corporation (GRNVAC1), Dendreon Corp (Provenge),BN ImmunoTherapeutics (PROSTVAC), GlobeImmune (Tarmogens), Advaxis, Inc(Lovaxin C), Accentia Biopharmaceuticals majority owned subsidiaryBiovest International [BiovaxID], GeneMax Corp (GMXX), (Apthera, Inc.(NeuVax).

Despite the potency and specificity of the immune system, vaccinationwith tumor antigens generally fails to eradicate cancer in mice andhumans (1,2). Currently, the most successful form of immunotherapy isadoptive cell therapy, which includes ex-vivo activation oftumor-infiltrating lymphocytes (TILs) and re-infusion of these cellsalong with high doses of cytokines. This approach is limited by cytokinetoxicity and by the limited range of tumors from which sufficient TILscan be obtained (melanoma) (3).

Bone marrow transplantation has become well established in the treatmentof malignant disorders. High-dose chemotherapy with hematopoietic stemcell support is widely used for most hematological malignancies, as wellas for some solid tumors. In light of recent developments in bloodprogenitor cell harvest, in particular, the availability of largenumbers of blood stem cells, mobilized by granulocyte colony-stimulatingfactor and collected by leukapheresis, it is possible to overcomehistocompatibility barriers in HLA-mismatched patients. Other recentdevelopments including but not limited to new methods for bloodprogenitor cells mobilization and ex vivo expansion of progenitor cellsand immune cells, the use of umbilical cord blood as an alternativesource of stem cells, and other molecular techniques, support aneffective treatment of cancer via autologous transplantation ofhematopoietic and immune cells.

In one aspect, the present invention provides a method for treatingcancer comprising: a) obtaining purified tumor cells; b) vaccinating adonor subject with the purified tumor cells; c) collecting immune cellsfrom the vaccinated donor; and d) transplanting the collected immunecells from the donor into a tumor-bearing recipient following total bodyirradiation of the recipient. In some embodiments, the method of thepresent invention combines vaccinating the donor with stimulated tumorcells and transplantation of hematopoietic and immune cells collectedfrom the donor to a tumor-bearing recipient. In some embodiments, thesubject method comprises autologous tumor vaccination followed byautologous transplantation of hematopoietic and immune cells, whereinthe subject is vaccinated with its own tumor cells, for example, tumorcells stimulated with one or more adjuvants, and then transplanted withits own hematopoietic and immune cells collected after tumorvaccination. The subject typically receives total body irradiationbefore transplantation of hematopoietic and immune cells.

In one example, the present invention shows that mice that havedeveloped disseminated tumors or bulky primary tumors established for 2weeks following inoculation with the CT26 colon carcinoma cells can becured, when treated with a combination of tumor vaccination andhematopoetic cell transplantation (HCT), without ex-vivo T cellactivation or use of TILs. Prior attempts at effective treatment byvaccination of mice with unmodified CT26 cells have failed duepresumably to the low immunogenicity of this tumor (4). Severalstrategies have been used to overcome this problem including vaccinationwith GM-CSF transfected CT26 cells as well as with altered ligands withheteroclitic activity (4-7). In the present invention, by combiningvaccination with HCT, a strong immune response to unmodified CT26 tumorcells is induced.

Tumor Cell Purification for Vaccination

In one aspect, the present invention provides a method for treatingcancer comprising: a) obtaining purified tumor cells; b) vaccinating adonor subject with the purified tumor cells; c) collecting immune cellsfrom the vaccinated donor; and d) transplanting the collected immunecells from the donor into a tumor-bearing recipient following total bodyirradiation of the recipient. In another aspect, the present inventionprovides a method for purifying tumor cells from vaccination comprising:a) obtaining a tumor tissue from a subject; b) making cell suspension ofthe tumor tissue; c) separating tumor cells from the cell suspension;and d) obtaining purified tumor cells with a purity of at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

In some embodiments, tumor cells are purified by Ficoll gradient. Ficollis a neutral, highly branched, high-mass, hydrophilic polysaccharidewhich dissolves readily in aqueous solutions. Ficoll radii range from2-7 nm. It is prepared by reaction of the polysaccharide withepichlorohydrin. Ficoll is part of Ficoll-Paque which is used in biologylaboratories to separate blood to its components (erythrocytes,leukocytes etc.) Ficoll-Paque is normally placed at the bottom of aconical tube, and blood is then slowly layered above Ficoll-Paque. Afterbeing centrifuged, the following layers will be visible in the conicaltube, from top to bottom: plasma and other constituents, a layer ofmono-nuclear cells called buffy coat (PBMC/MNC), Ficoll-Paque, anderythrocytes & granulocytes which should be present in pellet form. Thisseparation allows easy harvest of PBMC's. Note that some red blood celltrapping (presence of erythrocytes & granulocytes) may occur in the PBMCor Ficoll-Paque layer. Major blood clotting may sometimes occur in thePBMC layer. Ethylene diamine tetra-acetate (EDTA) and heparin arecommonly used in conjunction with Ficoll-Paque™ to prevent clotting.Ficoll can also be used to separate islets of Langerhans from pancreatictissue. The separated islets can then be used for transplantation intopatients with type 1 diabetes.

Separation of viable from non-viable human tumor cells can be performedby differential flotation on Ficoll-Hypaque specific density solution.In some embodiments, Ficoll-Hypaque is used to separate tumor cells fromnecrotic tissue. Ficoll gradient or filtration can also be used toseparate tumor cells from normal blood cells.

In some embodiments, tumor cells are purified by Percoll. Percoll is atool for efficient density separation. It is used for the isolation ofcells, organelles, and/or viruses by density centrifugation. Percollconsists of colloidal silica particles of 15-30 nm diameter (23% w/w inwater) which have been coated with polyvinylpyrrolidone (PVP). Percollis well suited for density gradient experiments because it possesses alow viscosity compared to alternatives, a low osmolarity and no toxicitytowards cells and their constituents.

In some embodiments, tumor cells are purified from other components inthe tumor tissue suspension based on their cell surface markers. Tumormarkers are substances that can be found in the body when cancer ispresent. They are most often found in the blood or urine, but they canalso be found in tumors and other tissue. They can be products of thecancer cells themselves, or made by the body in response to cancer orother conditions. Most tumor markers are proteins. There are manydifferent tumor markers. Some are seen only in a single type of cancer,while others can be found in many types of cancer. Examples of tumorcell surface markers that can be used to isolate tumor cells include butare not limited to prostate specific membrane antigen (PSMA) on prostatecancer cells, MAGE, GAGE and BAGE in ovarian cancer and melanomatissues, PLAC1 (PLACenta-specific 1) in human hepatocellular cancer(HCC) tissues, and epithelial tumor antigen (ETA), e.g. MUC1 on breastcancer cells.

The purity of tumor cells purified from the tumor tissue and suspensionby the subject methods of the present invention is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%. or more. The other components that are removed from the tumorcells during the purification process of the present invention mayinclude but are not limited to stromal cells, immunoregulatory cellssuch as regulatory T cells, myeloid suppressor cells, andimmunosuppressive cytokines.

In some embodiments, the purified tumor cells are irradiated prior tovaccination. For example, the tumor cells can be washed and irradiatedat 5,000-20,000 rads.

In some embodiments, the purified irradiated tumor cells are stimulatedwith an adjuvant to increase immunogenecity. An adjuvant is an agentthat may stimulate the immune system and increase the response to avaccine, without having any specific antigenic effect in itself. Animmunologic adjuvant is defined as any substance that acts toaccelerate, prolong, or enhance antigen-specific immune responses whenused in combination with specific vaccine antigens, and thus providingincreased immunity to a particular disease. Adjuvants accomplish thistask by mimicking specific sets of evolutionarily conserved molecules,so called PAMPs, which include liposomes, lipopolysaccharide (LPS),molecular cages for antigen, components of bacterial cell walls, andendocytosed nucleic acids such as double-stranded RNA (dsRNA),single-stranded DNA (ssDNA), and unmethylated CpGdinucleotide-containing DNA (Gavin A, et al. (2006) Science 314 (5807):1936-8). Because immune systems have evolved to recognize these specificantigenic moieties, the presence of an adjuvant in conjunction with thevaccine can greatly increase the innate immune response to the antigenby augmenting the activities of dendritic cells (DCs), lymphocytes, andmacrophages by mimicking a natural infection. Furthermore, becauseadjuvants are attenuated beyond any function of virulence, they poselittle or no independent threat to a host organism.

The ability of immune system to recognize molecules that are broadlyshared by pathogens is, in part, due to the presence of special Immunereceptors called TLRs that are expressed on leukocyte membranes. Thebinding of ligand—either in the form of adjuvant used in vaccinations orin the form of invasive moieties during times of natural infection—tothe TLR marks the key molecular events that ultimately lead to innateimmune responses and the development of antigen-specific acquiredimmunity (Medzhitov R, Preston-Hurlburt P, Janeway C (1997) Nature 388(6640): 394-7). Examples of adjuvant that can be used in the subjectmethods of the present invention include but are not limited to CpG,LPS, polyIC, imiquimod, and GM-CSF. Stimulation of tumor cells in thepresence of an adjuvant is well known to one skilled in the art.

Storing the Purified Tumor Cells

It may be advantageous to store purified tumor cells prior to, during,or after use of the cells for vaccination. For example, the purifiedtumor cells can be stored upon acquisition to facilitate transport, orto wait for the results of other analyses. In another embodiment,purified tumor cells are provided to physicians for appropriatetreatment of cancer. In another embodiment, purified tumor cells arestored while awaiting instructions from a physician or other medicalprofessional. In some cases, a portion of the purified tumor cells arestored while another portion of the purified tumor cells is furthermanipulated. Such manipulations can include but are not limited tomolecular profiling, cytological staining, gene or gene expressionproduct extraction, fixation, and examination.

The purified tumor cells may be placed in a suitable medium, excipient,solution, or container for short term or long term storage. Said storagemay require keeping the cells in a refrigerated, or frozen environment.The tumor cells may be quickly frozen prior to storage in a frozenenvironment. The frozen sample may be contacted with a suitablecryopreservation medium or compound including but not limited to:glycerol, ethylene glycol, sucrose, or glucose. A suitable medium,excipient, or solution may include but is not limited to: hanks saltsolution, saline, cellular growth medium, or water. The medium,excipient, or solution may or may not be sterile.

The medium, excipient, or solution may contain preservative agents tomaintain the sample in an adequate state for subsequent diagnostics ormanipulation, or to prevent coagulation. Said preservatives may includecitrate, ethylene diamine tetraacetic acid, sodium azide, or thimersol.The sample may be fixed prior to or during storage by any method knownto the art such as using glutaraldehyde, formaldehyde, or methanol. Thecontainer may be any container suitable for storage and or transport ofthe biological sample including but not limited to: a cup, a cup with alid, a tube, a sterile tube, a vacuum tube, a syringe, a bottle, amicroscope slide, or any other suitable container. The container may ormay not be sterile. In some cases, the sample may be stored in acommercial preparation suitable for storage of cells for subsequentcytological analysis such as but not limited to Cytyc ThinPrep,SurePath, or Monoprep.

Business Method

Also provided by the present invention is a business method of providingpurified tumor cells of the present invention to a third party. Asdescribed herein, the term customer or potential customer refers toindividuals or entities that may utilize methods or services of thetumor cell purification business. Potential customers for the tumor cellpurification methods and services described herein include for example,patients, subjects, physicians, cytological labs, health care providers,researchers, insurance companies, government entities such as Medicaid,employers, or any other entity interested in achieving more economicalor effective system for diagnosing, monitoring and treating cancer.

Such parties can utilize the purified tumor cells obtained from thetumor cell purification method of the present invention, for example, tovaccinate patients having such cancer for treatment.

Tumor Vaccination and Hematopoietic Cell Transplantation (HCT) as CancerTherapy

In some embodiments, the purified irradiated tumor cells are mixed Withadjuvant and injected into a subject for vaccination. In someembodiments, the subject is bearing a tumor. In some embodiments, thevaccination is autologous tumor vaccination wherein the tumor cells areinjected into the subject from whom the tumor cells were originallyobtained. The tumor cells can be obtained from a primary tumor, adisseminated tumor, or a metastatic tumor. In some embodiments, thetumor is a solid tumor. In some embodiments, tumor cells areadministered to the subject parenterally, including intramuscular,intraarterial, intrathecal, intradermal, intraperitoneal, intrasplenic,subcutaneous, and intravenous administration. In some embodiments theadjuvant is injected directly into the tumor.

After vaccination with the purified tumor cells of the presentinvention, peripheral blood immune cells are collected. In someembodiments, the immune cells are T lymphocytes, i.e. T cells. Theimmune cells can be collected several weeks after tumor cellvaccination, for example, peripheral blood T cells can be collected 6weeks after tumor vaccination. In some embodiments, hematopoieticprogenitor cells, such as CD34⁺ cells, are mobilized with granulocytecolony-stimulating factor (G-CSF). G-CSF is a colony-stimulating factorhormone. It is a glycoprotein, growth factor or cytokine produced by anumber of different tissues to stimulate the bone marrow to producegranulocytes and stem cells. G-CSF then stimulates the bone marrow torelease them into the blood. G-CSF is also a potent inducer of HSCsmobilization from the bone marrow into the bloodstream, although it hasbeen shown that it does not directly affect the hematopoieticprogenitors that are mobilized. Therefore, G-CSF is used to increase thenumber of hematopoietic stem cells in the blood of the donor beforecollection by leukapheresis for use in hematopoietic stem celltransplantation. It may also be given to the receiver, to compensate forconditioning regimens.

In some embodiments, CD34⁺ hematopoietic progenitor cells are enriched.CD34 molecule is a cluster of differentiation molecule present oncertain cells within the human body. It is a cell surface glycoproteinand functions as a cell-cell adhesion factor. It may also mediate theattachment of stem cells to bone marrow extracellular matrix or directlyto stromal cells. Known methods in the art can be used to enrich CD34⁺hematopoietic progenitor cells. For example, CD34+ cells may be isolatedfrom blood samples using immunomagnetic or immunofluorescent methods.Antibodies are used to quantify and purify hematopoietic progenitor stemcells for research and for clinical bone marrow transplantation. In oneembodiment, iso-osmolar Percoll density gradient is used to enrich CD34⁺cells. In another embodiment, an immunomagnetic separation techniqueusing anti-CD34 antibody or magnetic beads coated with anti-CD34antibody is used to enrich CD34⁺ cells.

In some embodiments, both CD34⁺ progenitor cells and peripheral T cellsare collected from the vaccinated subject for subsequent transplantationinto the tumor-bearing recipient. In some embodiments, suchtransplantation is autologous wherein the CD34⁺ progenitor cells andperipheral immune cells are collected from a vaccinated cancer patientand subsequently injected back into the same patient following totalbody irradiation of the patient. Transplantation is typically performedparenterally, for example, via intravenous infusion.

In some embodiments, the recipient receives a total body irradiation(TBI) prior to receiving hematopoietic and immune cell transplantation.In some embodiments, the patient receives TBI before autologoustransplantation. Total body irradiation (TBI) is a form of radiotherapyused primarily as part of the preparative regimen for hematopoietic stemcell (or bone marrow) transplantation. TBI involves irradiation of theentire body, though in modern practice the lungs are often partiallyshielded to lower the risk of radiation-induced lung injury. Total bodyirradiation in the setting of bone marrow transplantation serves todestroy or suppress the recipient's immune system, preventingimmunologic rejection of transplanted donor bone marrow or blood stemcells. Additionally, high doses of total body irradiation can eradicateresidual cancer cells in the transplant recipient, increasing thelikelihood that the transplant will be successful.

Doses of total body irradiation used in bone marrow transplantationtypically range from 10 to >12 Gy. For reference, a dose of 4.5 Gy isfatal in 50% of exposed individuals without aggressive medical care. Atthese doses, total body irradiation both destroys the patient's bonemarrow (allowing donor marrow to engraft) and kills residual cancercells. Non-myeloablative bone marrow transplantation uses lower doses oftotal body irradiation, typically about 2 Gy, which do not destroy thehost bone marrow but do suppress the host immune system sufficiently topromote donor engraftment.

In some embodiments, total body irradiation is fractionated. That is,the radiation is delivered in multiple small doses rather than one largedose. It has been demonstrated that delivering TBI through multiplesmaller doses resulted in lower toxicity and better outcomes thandelivering a single, large dose (Thomas E D, Buckner C D, Clift R A, etal. (1979) N. Engl. J. Med. 301 (11): 597-9). In other embodiments, TBIis delivered in one single dose.

Following total body irradiation, the recipient typically receiveshematopoietic and immune cell transplantation, in a preferredembodiment, autologous transplantation. Hematopoietic stem celltransplantation (HSCT) is the transplantation of blood stem cellsderived from the bone marrow or blood. Stem cell transplantation is amedical procedure in the fields of hematology and oncology, most oftenperformed for people with diseases of the blood, bone marrow, or certaintypes of cancer. With the availability of the stem cell growth factorsGM-CSF and G-CSF, most hematopoietic stem cell transplantationprocedures are now performed using stem cells collected from theperipheral blood, rather than from the bone marrow.

Autologous HSCT requires the extraction (apheresis) of hematopoieticstem cells (HSC) from the patient and storage of the harvested cells ina freezer. The patient is typically treated with high-dose chemotherapywith or without radiotherapy with the intention of eradicating thepatient's malignant cell population at the cost of partial or completebone marrow ablation (destruction of patient's bone marrow function togrow new blood cells). The patient's own stored stem cells are thenreturned to his/her body, where they replace destroyed tissue and resumethe patient's normal blood cell production. Autologous transplants havethe advantage of lower risk of infection during the immune-compromisedportion of the treatment since the recovery of immune function is rapid.Also, the incidence of patients experiencing rejection(graft-versus-host disease) is very rare due to the donor and recipientbeing the same individual. These advantages have established autologousHSCT as one of the standard second-line treatments for such diseases aslymphoma (Canellos, George (1997) The Oncologist 2 (3): 181-183).

In some embodiments, the subject method further comprises vaccinatingthe recipient with the purified tumor cells of the present inventionafter the transplantation. For example, the recipient can be vaccinatedwith the purified tumor cells one more time after having received thetransplantation.

Clinical Efficacy

Tumor growth and disease progression is monitored during and aftertreatment of cancer via the subject methods of the present invention.Clinical efficacy can be measured by any method known in the art. Insome embodiments, clinical efficacy of the subject treatment method isdetermined by measuring the clinical benefit rate (CBR).

The clinical benefit rate is measured by determining the sum of thepercentage of patients who are in complete remission (CR), the number ofpatients who are in partial remission (PR) and the number of patientshaving stable disease (SD) at a time point at least 6 months out fromthe end of therapy. The shorthand for this formula is CBR=CR+PR+SD≧6months. In some embodiments, CBR for the subject treatment method is atleast about 50%. In some embodiments, CBR for the subject treatmentmethod is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore.

In the present invention, the preclinical data show for the first timethat it is possible to eradicate primary, metastatic, or disseminatedsolid tumors by treating tumor bearing hosts with HCT containingsensitized T cells from vaccinated donors. While there is growingevidence that hematologic cancers can be effectively treated with acombination of tumor vaccination and HCT (15,16), the effect of suchtreatment on solid tumors has not been tested. Our outcome measure fortumor immunity in the present invention was eradication of the CT26 orMC38 colon tumors.

An important limitation of allogeneic HCT is the development of graftversus host disease (GVHD), which occurs in a severe form in about30-50% of humans who receive this therapy (19-21). GVHD is likely to beaggravated by vaccinating donors with tumors expressing hostalloantigens. By combining tumor vaccination of the donor with syngeneicHCT for the treatment of primary and metastatic colon cancer in mice,not only GVHD is avoided but also a potent and durable anti-tumorresponse is achieved. The results demonstrate a powerful synergy betweentumor vaccination and HCT in two genetically distinct mouse strains withtwo different colon tumors.

In the subject methods, a robust anti-tumor immune response can betransferred to tumor bearing mice without ex-vivo T cell expansion ortreatment of the mice with cytokines. Without being bound by any theory,the finding that CD4⁺ and CD8⁺ T cells needed to be included in thetransplant to achieve cures indicates that effective vaccinationrequires epitopes recognized by both types of T cells. Such epitopeswere lacking in a vaccine consisting of the immunodominant AH-1 peptideand CpG, which may explain why this vaccine was ineffective, in contrastto vaccines containing whole tumor cells, which are a source of multipleCD4 and CD8 epitopes. CD4⁺ T cells provide help to memory CD8⁺ T cellsby enhancing their immune potency, expansion, and persistence afterexposure to antigen (24).

Without being bound by any theory, the subject methods indicate thatirradiation of tumor bearing hosts was also required for tumor cures,and markedly augmented the expansion of transplanted T cells in thespleen and their infiltration into tumors. Since lethal irradiation wasconsiderably more effective than sublethal irradiation, hematopoeticstem cells had to be included in the transplants to rescue hosts frommarrow aplasia in the present invention. Previous studies indicate thatthe hematopoetic stem cells injected into irradiated mice not onlyprevented marrow aplasia, but also facilitated the expansion of CD8⁺ Tcells directed to melanoma tumor antigens by enhancing IL-7 and IL-15production (22).

In the present invention, we found that irradiation and HCT altered thebalance of T cell subsets infiltrating the tumors rather than simplydepleting T regulatory cells at the tumor site. Since CD4⁺CD25⁺FoxP3⁺Treg cells can suppress tumor immunity (14), and CD8⁺ effector memory Tcells can mediate tumor cell killing, the balance of the subsets wasdetermined in tumor bearing mice with or without HCT. Mice givenirradiation and HCT had a 10 fold higher ratio of CD8⁺ effector memory Tcells:Treg cells in the tumors as compared to control mice without HCT.Thus, the HCT procedure not only increases the absolute number of Tcells that infiltrated the tumors, but also favors the T cell subsetsthat kill tumor cells versus the subset that suppresses tumor immunity.

Tumor vaccination without HCT was not effective against establishedtumors. However, the subject methods demonstrate that vaccination oftumor-bearing animals provided long-term, transferable immunity, whichcan be enhanced by HCT. These data suggest that patients whose primarytumors are resected but remain at high risk for relapse, can benefitfrom early vaccination combined with HCT in the event of relapse.

Methods of Treatment: Anti-Cancer Therapy

One aspect of the present invention relates to a method for treatingcancer comprising: a) obtaining purified tumor cells; b) vaccinating adonor subject with the purified tumor cells; c) collecting immune cellsfrom the vaccinated donor; and d) transplanting the collected immunecells from the donor into a tumor-bearing recipient following total bodyirradiation of the recipient. The term “subject” as used herein includeshumans as well as other mammals. The term “treating” as used hereinincludes achieving a therapeutic benefit and/or a prophylactic benefit.By therapeutic benefit is meant eradication or amelioration of thecancer. Also, a therapeutic benefit is achieved with the eradication oramelioration of one or more of the physiological symptoms associatedwith cancer such that an improvement is observed in the animal subject,notwithstanding the fact that the animal subject may still be afflictedwith that cancer.

The types of cancer that can be treated using the subject methods of thepresent invention include but are not limited to adrenal corticalcancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer,bone cancer, bone metastasis, brain cancers, central nervous system(CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer,cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectumcancer, endometrial cancer, esophagus cancer, Ewing's family of tumors(e.g. Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinalcarcinoid tumors, gastrointestinal stromal tumors, gestationaltrophoblastic disease, hairy cell leukemia, Hodgkin's lymphoma, Kaposi'ssarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acutelymphocytic leukemia, acute myeloid leukemia, children's leukemia,chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer,lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breastcancer, malignant mesothelioma, multiple myeloma, myelodysplasticsyndrome, myeloproliferative disorders, nasal cavity and paranasalcancer, nasopharyngeal cancer, neuroblastoma, oral cavity andoropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,penile cancer, pituitary tumor, prostate cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma skin cancer,non-melanoma skin cancers, stomach cancer, testicular cancer, thymuscancer, thyroid cancer, uterine cancer (e.g. uterine sarcoma),transitional cell carcinoma, vaginal cancer, vulvar cancer,mesothelioma, squarnous cell or epidermoid carcinoma, bronchial adenoma,choriocarinoma, head and neck cancers, teratocarcinoma, or Waldenstrom'smacroglobulinemia.

In a preferred embodiment, the subject method is used to treat a solidtumor, for example, colorectal cancer, lung cancer, liver cancer, breastcancer, prostate cancer, ovarian cancer or pancreatic cancer.

Combination Therapy

In some embodiments, the subject method further comprises administeringto a subject in need thereof an anti-tumor agent, or a pharmaceuticallyacceptable salt or prodrug thereof. In some embodiments, the anti-tumoragents include but are not limited to antitumor alkylating agents,antitumor antimetabolites, antitumor antibiotics, plant-derivedantitumor agents, antitumor organoplatinum compounds, antitumorcampthotecin derivatives, antitumor tyrosine kinase inhibitors,monoclonal antibodies, interferons, biological response modifiers, andother agents having antitumor activities, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the subject method further comprises treating asubject in need thereof one or more of the following therapies incombination with the subject method disclosed herein.

Antineoplastic Chemotherapeutic Agents

Suitable antineoplastic anti-tumor agents to be used in the presentinvention include, but are not limited to, alkylating agents,antimetabolites, natural antineoplastic agents, hormonal antineoplasticagents, angiogenesis inhibitors, differentiating reagents, RNAinhibitors, antibodies or immunotherapeutic agents, gene therapy agents,small molecule enzymatic inhibitors, biological response modifiers, andanti-metastatic agents.

Alkylating Agents

Alkylating agents are known to act through the alkylation ofmacromolecules such as the DNA of cancer cells, and are usually strongelectrophiles. This activity can disrupt DNA synthesis and celldivision. Examples of alkylating reagents suitable for use hereininclude nitrogen mustards and their analogues and derivatives including,cyclophosphamide, ifosfamide, chlorambucil, estramustine,mechlorethamine hydrochloride, melphalan, and uracil mustard. Otherexamples of alkylating agents include alkyl sulfonates (e.g. busulfan),nitrosoureas (e.g. carmustine, lomustine, and streptozocin), triazenes(e.g. dacarbazine and temozolomide), ethylenimines/methylmelamines (e.g.altretamine and thiotepa), and methylhydrazine derivatives (e.g.procarbazine). Included in the alkylating agent group are thealkylating-like platinum-containing drugs comprising carboplatin,cisplatin, and oxaliplatin.

Antimetabolites

Antimetabolic antineoplastic agents structurally resemble naturalmetabolites, and are involved in normal metabolic processes of cancercells such as the synthesis of nucleic acids and proteins. They differenough from the natural metabolites so that they interfere with themetabolic processes of cancer cells. Suitable antimetabolicantineoplastic agents to be used in the present invention can beclassified according to the metabolic process they affect, and caninclude, but are not limited to, analogues and derivatives of folicacid, pyrimidines, purines, and cytidine. Members of the folic acidgroup of agents suitable for use herein include, but are not limited to,methotrexate (amethopterin), pemetrexed and their analogues andderivatives. Pyrimidine agents suitable for use herein include, but arenot limited to, cytarabine, floxuridine, fluorouracil (5-fluorouracil),capecitabine, gemcitabine, and their analogues and derivatives. Purineagents suitable for use herein include, but are not limited to,mercaptopurine (6-mercaptopurine), pentostatin, thioguanine, cladribine,and their analogues and derivatives. Cytidine agents suitable for useherein include, but are not limited to, cytarabine (cytosinearabinodside), azacitidine (5-azacytidine) and their analogues andderivatives.

Natural Antineoplastic Agents

Natural antineoplastic agents comprise antimitotic agents, antibioticantineoplastic agents, camptothecin analogues, and enzymes. Antimitoticagents suitable for use herein include, but are not limited to, vincaalkaloids like vinblastine, vincristine, vindesine, vinorelbine, andtheir analogues and derivatives. They are derived from the Madagascarperiwinkle plant and are usually cell cycle-specific for the M phase,binding to tubulin in the microtubules of cancer cells. Otherantimitotic agents suitable for use herein are the podophyllotoxins,which include, but are not limited to etoposide, teniposide, and theiranalogues and derivatives. These reagents predominantly target the G2and late S phase of the cell cycle.

Also included among the natural antineoplastic agents are the antibioticantineoplastic agents: Antibiotic antineoplastic agents areantimicrobial drugs that have anti-tumor properties usually throughinteracting with cancer cell DNA. Antibiotic antineoplastic agentssuitable for use herein include, but are not limited to, belomycin,dactinomycin, doxorubicin, idarubicin, epirubicin, mitomycin,mitoxantrone, pentostatin, plicamycin, and their analogues andderivatives.

The natural antineoplastic agent classification also includescamptothecin analogues and derivatives which are suitable for use hereinand include camptothecin, topotecan, and irinotecan. These agents actprimarily by targeting the nuclear enzyme topoisomerase I. Anothersubclass under the natural antineoplastic agents is the enzyme,L-asparaginase and its variants. L-asparaginase acts by depriving somecancer cells of L-asparagine by catalyzing the hydrolysis of circulatingasparagine to aspartic acid and ammonia.

Hormonal Antineoplastic Agents

Hormonal antineoplastic agents act predominantly on hormone-dependentcancer cells associated with prostate tissue, breast tissue, endometrialtissue, ovarian tissue, lymphoma, and leukemia. Such tissues may beresponsive to and dependent upon such classes of agents asglucocorticoids, progestins, estrogens, and androgens. Both analoguesand derivatives that are agonists or antagonists are suitable for use inthe present invention to treat tumors. Examples of glucocorticoidagonists/antagonists suitable for use herein are dexamethasone,cortisol, corticosterone, prednisone, mifepristone (RU486), theiranalogues and derivatives. The progestin agonist/antagonist subclass ofagents suitable for use herein includes, but is not limited to,hydroxyprogesterone, medroxyprogesterone, megestrol acetate,mifepristone (RU486), ZK98299, their analogues and derivatives. Examplesfrom the estrogen agonist/antagonist subclass of agents suitable for useherein include, but are not limited to, estrogen, tamoxifen, toremifene,RU58668, SR16234, ZD164384, ZK191703, fulvestrant, their analogues andderivatives. Examples of aromatase inhibitors suitable for use herein,which inhibit estrogen production, include, but are not limited to,androstenedione, formestane, exemestane, aminoglutethimide, anastrozole,letrozole, their analogues and derivatives. Examples from the androgenagonist/antagonist subclass of agents suitable for use herein include,but are not limited to, testosterone, dihydrotestosterone,fluoxymesterone, testolactone, testosterone enanthate, testosteronepropionate, gonadotropin-releasing hormone agonists/antagonists (e.g.leuprolide, goserelin, triptorelin, buserelin), diethylstilbestrol,abarelix, cyproterone, flutamide, nilutamide, bicalutamide, theiranalogues and derivatives.

Angiogenesis Inhibitors

Angiogenesis inhibitors work by inhibiting the vascularization oftumors. Angiogenesis inhibitors encompass a wide variety of agentsincluding small molecule agents, antibody agents, and agents that targetRNA function. Examples of angiogenesis inhibitors suitable for useherein include, but are not limited to, ranibizumab, bevacizumab,SU11248, PTK787, ZK222584, CEP-7055, angiozyme, dalteparin, thalidomide,suramin, CC-5013, combretastatin A4 Phosphate, LY317615, soyisoflavones, AE-941, interferon alpha, PTK787/ZK 222584, ZD6474, EMD121974, ZD6474, BAY 543-9006, celecoxib, halofuginone hydrobromide,bevacizumab, their analogues, variants, or derivatives.

Differentiating Reagents

Differentiating agents inhibit tumor growth through mechanisms thatinduce cancer cells to differentiate. One such subclass of these agentssuitable for use herein includes, but is not limited to, vitamin Aanalogues or retinoids, and peroxisome prolifeiator-activated receptoragonists (PPARs). Retinoids suitable for use herein include, but are notlimited to, vitamin A, vitamin A aldehyde (retinal), retinoic acid,fenretinide, 9-cis-retinoid acid, 13-cis-retinoid acid,all-trans-retinoic acid, isotretinoin, tretinoin, retinyl palmitate,their analogues and derivatives. Agonists of PPARs suitable for useherein include, but are not limited to, troglitazone, ciglitazone,tesaglitazar, their analogues and derivatives.

Antibodies/Immunotherapeutic Agents

Antibody agents bind targets selectively expressed in cancer cells andcan either utilize a conjugate to kill the cell associated with thetarget, or elicit the body's immune response to destroy the cancercells. Immunotherapeutic agents can either be comprised of polyclonal ormonoclonal antibodies. The antibodies may be comprised of non-humananimal (e.g. mouse) and human components, or be comprised of entirelyhuman components (“humanized antibodies”). Examples of monoclonalimmunotherapeutic agents suitable for use herein include, but are notlimited to, rituximab, tosibtumomab, ibritumomab which target the CD-20protein. Other examples suitable for use herein include trastuzumab,edrecolomab, bevacizumab, cetuximab, carcinoembryonic antigenantibodies, gemtuzumab, alemtuzumab, mapatumumab, panitumumab, EMD72000, TheraCIM hR3, 2C4, HGS-TR2J, and HGS-ETR2.

Gene Therapy Agents

Gene therapy agents insert copies of genes into a specific set of apatient's cells, and can target both cancer and non-cancer cells. Thegoal of gene therapy can be to replace altered genes with functionalgenes, to stimulate a patient's immune response to cancer, to makecancer cells more sensitive to chemotherapy, to place “suicide” genesinto cancer cells, or to inhibit angiogenesis. Genes may be delivered totarget cells using viruses, liposomes, or other carriers or vectors.This may be done by injecting the gene-carrier composition into thepatient directly, or ex vivo, with infected cells being introduced backinto a patient. Such compositions are suitable for use in the presentinvention.

Nanotherapy

Nanometer-sized particles have novel optical, electronic, and structuralproperties that are not available from either individual molecules orbulk solids. When linked with tumor-targeting moieties, such astumor-specific ligands or monoclonal antibodies, these nanoparticles canbe used to target cancer-specific receptors, tumor antigens(biomarkers), and tumor vasculatures with high affinity and precision.The formulation and manufacturing process for cancer nanotherapy isdisclosed in patent U.S. Pat. No. 7,179,484, and article M. N. Khalid,P. Simard, D. Hoarau, A. Dragomir, J. Leroux, Long CirculatingPoly(Ethylene Glycol)Decorated Lipid Nanocapsules Deliver Docetaxel toSolid Tumors, Pharmaceutical Research, 23(4), 2006, all of which areherein incorporated by reference in their entireties.

RNA Therapy

RNA including but not limited to siRNA, shRNA, microRNA may be used tomodulate gene expression and treat cancers. Double strandedoligonucleotides are formed by the assembly of two distinctoligonucleotide sequences where the oligonucleotide sequence of onestrand is complementary to the oligonucleotide sequence of the secondstrand; such double stranded oligonucleotides are generally assembledfrom two separate oligonucleotides (e.g., siRNA), or from a singlemolecule that folds on itself to form a double stranded structure (e.g.,shRNA or short hairpin RNA). These double stranded oligonucleotidesknown in the art all have a common feature in that each strand of theduplex has a distinct nucleotide sequence, wherein only one nucleotidesequence region (guide sequence or the antisense sequence) hascomplementarity to a target nucleic acid sequence and the other strand(sense sequence) comprises nucleotide sequence that is homologous to thetarget nucleic acid sequence.

MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23nucleotides in length, which regulate gene expression miRNAs are encodedby genes that are transcribed from DNA but not translated into protein(non-coding RNA); instead they are processed from primary transcriptsknown as pri-miRNA to short stem-loop structures called pre-miRNA andfinally to functional miRNA. Mature miRNA molecules are partiallycomplementary to one or more messenger RNA (mRNA) molecules, and theirmain function is to downregulate gene expression.

Certain RNA inhibiting agents may be utilized to inhibit the expressionor translation of messenger RNA (“mRNA”) that is associated with acancer phenotype. Examples of such agents suitable for use hereininclude, but are not limited to, short interfering RNA (“siRNA”),ribozymes, and antisense oligonucleotides. Specific examples of RNAinhibiting agents suitable for use herein include, but are not limitedto, Cand5, Sirna-027, fomivirsen, and angiozyme.

Small Molecule Enzymatic Inhibitors

Certain small molecule therapeutic agents are able to target thetyrosine kinase enzymatic activity or downstream signal transductionsignals of certain cell receptors such as epidermal growth factorreceptor (“EGFR”) or vascular endothelial growth factor receptor(“VEGFR”). Such targeting by small molecule therapeutics can result inanti-cancer effects. Examples of such agents suitable for use hereininclude, but are not limited to, imatinib, gefitinib, erlotinib,lapatinib, canertinib, ZD6474, sorafenib (BAY 43-9006), ERB-569, andtheir analogues and derivatives.

Biological Response Modifiers

Certain protein or small molecule agents can be used in anti-cancertherapy through either direct anti-tumor effects or through indirecteffects. Examples of direct-acting agents suitable for use hereininclude, but are not limited to, differentiating reagents such asretinoids and retinoid derivatives. Indirect-acting agents suitable foruse herein include, but are not limited to, agents that modify orenhance the immune or other systems such as interferons, interleukins,hematopoietic growth factors (e.g. erythropoietin), and antibodies(monoclonal and polyclonal).

Anti-Metastatic Agents

The process whereby cancer cells spread from the site of the originaltumor to other locations around the body is termed cancer metastasis.Certain agents have anti-metastatic properties, designed to inhibit thespread of cancer cells. Examples of such agents suitable for use hereininclude, but are not limited to, marimastat, bevacizumab, trastuzumab,rituximab, erlotinib, MMI-166,GRN163L, hunter-killer peptides, tissueinhibitors of metalloproteinases (TIMPs), their analogues, derivativesand variants.

Chemopreventative Agents

Certain pharmaceutical agents can be used to prevent initial occurrencesof cancer, or to prevent recurrence or metastasis. In some embodiments,treatment of cancer with the subject methods is accompanied with the useof chemopreventative agents. Examples of chemopreventative agentssuitable for use herein include, but are not limited to, tamoxifen,raloxifene, tibolone, bisphosphonate, ibandronate, estrogen receptormodulators, aromatase inhibitors (letrozole, anastrozole), luteinizinghormone-releasing hormone agonists, goserelin, vitamin A, retinal,retinoic acid, fenretinide, 9-cis-retinoid acid, 13-cis-retinoid acid,all-trans-retinoic acid, isotretinoin, tretinoid, vitamin B6, vitaminB12, vitamin C, vitamin D, vitamin E, cyclooxygenase inhibitors,non-steroidal anti-inflammatory drugs (NSAIDs), aspirin, ibuprofen,celecoxib, polyphenols, polyphenol E, green tea extract, folic acid,glucaric acid, interferon-alpha, anethole dithiolethione, zinc,pyridoxine, finasteride, doxazosin, selenium, indole-3-carbinal,alpha-difluoromethylomithine, carotenoids, beta-carotene, lycopene,antioxidants, coenzyme Q10, flavonoids, quercetin, curcumin, catechins,epigallocatechin gallate, N-acetylcysteine, indole-3-carbinol, inositolhexaphosphate, isoflavones, glucanic acid, rosemary, soy, saw palmetto,and calcium. An additional example of chemopreventative agents suitablefor use in the present invention is cancer vaccines. These can becreated through immunizing a patient with all or part of a cancer celltype that is targeted by the vaccination process.

Side-Effect Limiting Agents

In some embodiments, treatment of cancer with the subject methods isaccompanied by administration of pharmaceutical agents that canalleviate the side effects produced by the antineoplastic agents. Suchagents suitable for use herein include, but are not limited to,anti-emetics, anti-mucositis agents, pain management agents, infectioncontrol agents, and anti-anemia/anti-thrombocytopenia agents. Examplesof anti-emetics suitable for use herein include, but are not limited to,5-hydroxytryptamine 3 receptor antagonists, metoclopramide, steroids,lorazepam, ondansetron, cannabinoids, their analogues and derivatives.Examples of anti-mucositis agents suitable for use herein include, butare not limited to, palifermin (keratinocyte growth factor),glucagon-like peptide-2, teduglutide, L-glutamine, amifostin, andfibroblast growth factor 20. Examples of pain management agents suitablefor use herein include, but are not limited to, opioids, opiates, andnon-steroidal anti-inflammatory compounds. Examples of agents used forcontrol of infection suitable for use herein include, but are notlimited to, antibacterials such as aminoglycosides, penicillins,cephalosporins, tetracyclines, clindamycin, lincomycin, macrolides,vancomycin, carbapenems, monobactams, fluoroquinolones, sulfonamides,nitrofurantoin, their analogues and derivatives. Examples of agents thatcan treat anemia or thrombocytopenia associated with chemotherapysuitable for use herein include, but are not limited to, erythropoietin,and thrombopoietin.

EXAMPLES

The following materials and methods apply to Examples 1-6.

Wild-type male BALB/c (H-2^(d)) mice, male BALB/c Rag2^(−/−) mice,wild-type male DBA2/J (H-2^(d)) mice, and wild-type female C57BL/6 micewere purchased from Jackson Laboratories (Bar Harbor, Me., USA). Micewere 5-8 weeks old. The Stanford University Committee on Animal Welfare(Administration Panel of Laboratory Animal Care) approved all mouseprotocols used in this study.

The CT26 cell line (an N-nitro-N-methylurethane-induced BALB/c murinecolon carcinoma) was purchased from ATCC (Manassas, Va.). The MC38 cellline (dimethyl-hydrazine induced C57BL/6 colon adenocarcinoma) waskindly provided by Dr. David Bartlett of the University of Pittsburgh(8). Cell lines were maintained in RPMI-1640 complete mediumsupplemented with 10% fetal calf serum, L-glutamine, 2 mercaptoethanol,streptomycin and penicillin. For intrasplenic injection, animals wereanesthetized with ketamine/xylazine. Laparotomy was performed, and 5×10⁵CT26 cells were injected in the spleen. Abdominal wall was closed withsurgical sutures.

In terms of vaccination, five-week-old male BALB/c mice were immunizedby subcutaneous injection of 1x106 irradiated (10,000cGy) CT26 cells and30 μg of CpG. Five-week-old female C57BL/6 mice were immunized bysubcutaneous injection of 1x106 irradiated (10,000cGy) MC38 cells and 30μg of CpG. AH-1 peptide (300 μg per vaccination) used in this study wasobtained from Sigma-Genosys. The peptide was >95% pure as indicated byanalytical HPLC. Lyophilized peptide was diluted in DMSO and stored at−20° C. until use. Oligonucleotide containing unmethylated CG motifs(CpG) (TCCATGACGTTCCTGACGTT (SEQ ID NO: 1)) was synthesized andphosphorothioate-stabilized by Oligos, Etc. The oligonucleotide wasreconstituted in sterile pyrogen-free water and then diluted in PBS forin vivo injections. 30 μg of ultrapure LPS (Invivogen) was used in someexperiments instead of CpG.

Irradiation was performed with a Philips X-ray unit (200 kV, 10 mA;Philips Electronic Instruments Inc., Rahway, N.J., USA) at a rate of 84cGy/min with a 0.5 mm Cu filter.

For donor cell preparation, single cell suspensions of bone marrow andspleen prepared according to previously described procedures (9). Somesamples were enriched either for CD4+ cells, CD8+ T cells or Thy1.2+cells with anti-CD4, anti-CD8 magnetic microbeads (Miltenyi Biotech) oranti-Thy1.2-biotin monoclonal antibodies (mAb) (5a-8; Caltag,Burlingame, Calif.) and streptavidin-magnetic beads (Miltenyi Biotech)respectively using the MidiMACS system (Miltenyi Biotech, Auburn,Calif.). Enriched cells were stained with anti-TCR-allophycocyanin (APC)and anti-CD4 or anti-CD8-fluorescein isothiocyanate (FITC) mAbs to checkfor purity, and preparations were uniformly at least 95% pure.

Purified HSCs were obtained by modification of the methods described bySpangrude et all (10). Thy-1lolin-/loSca-1+ c-Kit+ cells were sorted ona dual laser FACS (Becton Dickinson, Mountain View, Calif.) madeavailable through the FACS shared-user group at Stanford Universityusing FlowJo software (TreeStar, Ashland, Oreg.) for data analysis.After sorting cells were checked by FACS reanalysis and determined tobe >99% pure.

For histopathologic analysis, animals were killed when moribund as perStanford Animal Welfare protocol guidelines, or at 100 days aftertransplantation if they survived without morbidity. Histopathologicalspecimens were obtained from lungs and livers of hosts. Tissues werefixed in 10% formalin, stained with hematoxylin and eosin and imageswere obtained using an Eclipse E1000M microscope (Nikon, Melville, N.Y.,USA) as described before (11).

For analysis of donor cell accumulation in host spleens and tumornodules, single-cell suspensions were prepared from spleens and tumornodules of BALB/c recipients. The following reagents were used for flowcytometric analysis: unconjugated anti-CD16/32 (2.4G2 BD Biosciences),anti-CD4-FITC (RM4-5 BD Biosciences), anti-TCR-APC (H57-597 BDBiosciences), anti-CD8-APC-Cy7, (53-6.7 BD Biosciences), anti-Thy1.1PE-Cy7 (HIS51, eBioscience), anti-Thy1.2-biotin (5a-8; Caltag) mAbs, andstreptavidin-PE (SAv-phycoerythin, BecktonDickenson). All stainings wereperformed in PBS/1% calf serum in the presence of purified anti-CD16/32mAbs.

For statistical analysis, Kaplan-Meier survival curves were generatedusing Prism software (SAS Institute Inc., Cary, N.C., USA), andstatistical differences were analyzed using the log-rank (Mantel-Cox)test. Statistical significance in differences between mean percentage ofdonor cells in host spleens and tumors was analyzed using the two-tailedStudent's t-test of means. For all tests, P<0.05 was consideredsignificant.

Example 1 Hematopoietic Cell Transplantation (HCT) from VaccinatedBALB/c Donor Mice Cures Established CT26 Colon Tumors

FIG. 1A shows the experimental scheme, which uses HCT fromtumor-vaccinated donors to treat CT26 colon tumors in syngeneic mice. Inall instances, normal BALB/c donor mice were vaccinated subcutaneously(s.c.). with 10⁶ irradiated CT26 tumor cells mixed with 30 μg CpG, anadjuvant that stimulates antigen presenting cell via TLR-9 (12,13).After 90 days, spleen and bone marrow cells were harvested, andtransplanted intravenously (i.v.) into tumor-bearing BALB/c host micefollowing a single dose of total body irradiation (TBI). Seven daysprior to TBI, hosts had been given live tumor cells via s.c. (2.5×10⁴),i.v. (2×10⁵) or intrasplenic (5×10⁵) routes. FIG. 1B shows theprogressive growth of s.c. tumors in all untreated mice. Similarly,tumor bearing recipients of 50×10⁶ bone marrow cells and 60×10⁶ spleencells from unvaccinated donors had uniformly progressive tumors (FIG.1C). In contrast, after HCT from vaccinated donors, tumor bearing micedisplayed a steady regression of tumor volume over a 100 day observationperiod (FIG. 1D), which remained stable until the end of study (day 180;data not shown). Shortening the time interval between immunization ofthe donor and harvesting the graft from 90 to 14 days, but not to 50days, resulted in lower anti-tumor effect (p=0.005 and p=0.3,respectively; log rank test; FIG. 1E). Omission of CpG from the donorvaccine resulted in a further loss of efficacy (p=0.01), and only 20% ofhosts survived 100 days.

The same HCT strategy was also successful in recipients given tumorcells by i.v. administration. By day 7, tumor cells had disseminatedinto the lungs and formed multiple tumor clusters (FIG. 1H). By day 20all untreated control mice succumbed to progressive disease with large,nearly confluent tumor nodules (FIGS. 1F and 1H). In contrast,recipients of HCT from vaccinated donors all survived at least 100 days,with no histologic evidence of residual tumor (FIG. 1H). Accordingly,improvement of survival was significant as compared to untreated mice(p<0.01) (FIG. 1F). When tumor cells were injected into the spleen, byday 7 tumor nodules became established in the parenchyma of the liver(FIG. 1I), and by day 14 there was evidence of blood vessel invasion(arrows, FIG. 1I). All untreated animals died by day 30 (FIG. 1G) withmultiple visible, as well as microscopic, tumors. Treated mice survivedbeyond day 100 (FIG. 1G), easily exceeding the survival of untreatedmice (p=0.001). The liver of treated mice displayed no abnormalities andalso no histologic evidence of residual tumor at day 60 (FIG. 1I). HCTfrom vaccinated donors also cured peritoneal carcinomatosis, which hadbeen created by intraperitoneal injection of 5×10⁶ tumor cells and whichdisplayed multiple peritoneal nodules and ascites by the time oftransplant (data not shown). All untreated mice died by day 20, and alltransplanted mice survived at least 100 days without any peritonealtumor growth.

Example 2 Vaccination and HCT Induces Long-Term Anti-Tumor Immunity

To assess the duration of effect of vaccination combined with HCT,“cured” animals from the experiment in FIG. 1D were challenged with2.5×10⁴ live tumor cells at day 120 as shown in the experimental schemein FIG. 2A. The results show that these animals were completelyprotected and survived for at least 100 days (FIG. 2A). Moreover,harvesting of spleen and bone marrow cells from “cured” recipients atday 180 after HCT, and secondary transfer resulted in 80% of the newrecipients surviving for more than 100 days (FIG. 2B). At least 100 dayslater those secondary recipients were used as donors for another HCTinto irradiated tumor-bearing tertiary hosts which was also effectivesince all tertiary hosts survived more than 100 days (FIG. 2B). Thus,anti-tumor immunity generated by a single vaccination could eradicatetumors 370 days later (FIG. 2B).

Example 3 Tumor Eradication Requires Lethal Irradiation of Hosts, aswell as Transferof CD4⁺ and CD8⁺ T Cells from Vaccinated Donors

FIG. 3A illustrates the significant role of the host conditioningregimen in our vaccine strategy. All tumor bearing recipients of bonemarrow and splenocytes from vaccinated donors were cured whenconditioned with myeloablative TBI (800 cGy). In contrast, only 60% ofhosts survived 100 days with a non-myeloablative radiation dose (450cGy) (p<0.05), while none survived more than 40 days without irradiation(p<0.0001). Radiation causes lymphodepletion which might deplete hostregulatory T cells that suppress anti-tumor immune responses. In orderto test whether radiation mediates tumor eradication through such amechanism, we studied unirradiated tumor-bearing RAG2^(−/−) recipientsthat lack T cells. Rag 2^(−/−) BALB/c hosts were given myeloablativeradiation or no radiation immediately before transplantation of cellsfrom vaccinated donors. FIG. 3B shows that although there was asignificant delay in mortality in non-irradiated Rag 2−/− mice ascompared to non-irradiated wild type mice (p=0.001), all mice died byday 65. Conditioning of the Rag 2^(−/−) mice with 800 cGy resulted insignificant improvement in survival as compared to the non-irradiatedmice (p=0.001), and all hosts survived at least 100 days (FIG. 3B).

When IFNγ^(−/−) BALB/c mice were vaccinated and used as bone marrow andsplenocyte donors, the survival of hosts was decreased as compared towild type syngeneic donors (p=0.004) (FIG. 2C). Likewise, survival wasreduced in all five mice given grafts from vaccinated MHC-matched, minorantigen-mismatched DBA/2J donors (H-2^(d)) (p<0.001) (FIG. 3C). While4/5 animals had progressive tumor growth, one mouse displayed tumorregression, but succumbed due to GVHD. Substitution of whole tumor cellswith the tumor-associated immunodominant AH-1 peptide (4) forvaccination of donors resulted in decreased survival (p<0.0001) (FIG.3C). Use of lipopolysaccharide (30 μg) as an adjuvant for vaccinationwas just as effective as CpG, based on survival of hosts post-HCT(p=1.0) (FIG. 3C). Grafts from vaccinated donors consisting of bonemarrow or FACS-purified c-kit⁺Sca1^(high)lin⁻hematopoietic stem cells(HSC) failed to prevent tumor progression (FIG. 3D). Addition of 5×10⁶splenic T cells to the HSCs increased survival (p=0.008) such that themajority of hosts were alive at 100 days without detectable tumors (FIG.3D). When HSC grafts were supplemented with both CD4⁺ (3.5×10⁶) plusCD8⁺ T cells (1.8×10⁶), survival was improved (p<0.008) as compared tosupplementation with CD4⁺ or CD8⁺ T cells only (FIG. 3E).

The utility of HCT from vaccinated donors was further validated instudies of another colon cancer, MC38, which grows only in C57BL/6(H-2^(b)) mice (8). For these experiments, donor mice were vaccinatedwith 1×10⁶ MC38 tumor cells (FIG. 3F). Again, while syngeneic recipientsof grafts from vaccinated donors were cured, recipients of transplantsfrom unvaccinated donors did not survive beyond 35 days (p<0.0001). HCTfrom tumor-vaccinated donors could also significantly improve survivalof animals with large (>10 mm) tumors established for 15 days (p=0.0005)(FIG. 3G). After 100 days, 60% of treated animals were completely tumorfree.

Example 4 Analysis of Donor T Cells in Host Tumors and Spleens afterTransplantation: Irradiation Promotes T Cell Accumulation in Tumors

To delineate the donor-derived cell populations involved in theanti-tumor response, bone marrow and spleen cells were transplanted fromBALB/c Thy1.1 donors into tumor bearing BALB/c Thy 1.2 hosts, asdepicted in FIG. 4A. To assure that there would be sufficient donorcells for analysis at day 28, HCT was performed in animals bearingtumors that had been established for 14 instead of 7 days. In irradiatedrecipients of vaccinated donor grafts approximately 70% of T cellsinfiltrating the tumors were of donor origin (FIGS. 4B and 4C), whiledonor T cells accounted for <2% of tumoral T cells when hosts were notirradiated (p<0.001). A similar facilitation of donor cell accumulationin the spleen was observed in irradiated versus non-irradiated hosts(p<0.01) (FIGS. 4B and 4C). Differences in accumulation of total T cellsfrom vaccinated and unvaccinated donors were not significant (p>0.05).However, the majority of tumor T cells from vaccinated donors were CD8⁺,whereas most of the tumoral T cells from unvaccinated donors were CD4⁺(p=0.01) (FIGS. 4D and 4E). CD4⁺ T cells were in the majority in thespleens with both vaccinated and unvaccinated donors (p<0.001).

Example 5 HCT Alters the Balance between Regulatory and Effector Cellsat the Tumor Site

Previous studies have shown that CD4⁺CD25⁺FoxP3⁺ Treg cells can suppresstumor immunity (14). Moreover, this suppression was mediated at thetumor site and was lost after intra-tumoral depletion of Tregs (14). Itis shown above (FIG. 3B) that conditioning and HCT was required to curetumors in RAG2^(−/−) mice lacking T regulatory cells. Thus, therequirement for irradiation is not based on host Treg depletion.

However, regulatory T cells of donor or host origin may be capable ofinfiltrating tumors when wild-type hosts are used. Host and donor T cellsubsets infiltrating CT26 subcutaneous tumor nodules were examined inwild-type BALB/c mice before and after HCT, and in controls without HCTas shown in the experimental scheme in FIG. 5A. Control Thy1.2 micegiven CT26 cells subcutaneously were euthanized 14 or 28 days later, andsingle cell suspensions from tumors were analyzed for tumor infiltratingT lymphocytes (TIL) subsets.

Experimental mice were lethally irradiated and given HCT from vaccinatedThy 1.1 donors after 14 days of tumor growth, and tumor cell suspensionwere analyzed 14 days after HCT. FIG. 5B shows the representativestaining patterns for CD4⁺ and CD8⁺ T cells in cell suspensions usinggated Thy1.2⁺ T cells from control mice and gated Thy1.1⁺ from micegiven HCT at 28 days after the subcutaneous injection of tumor cells (14days after HCT).

Whereas CD8⁺ and CD4⁺ cells accounted for about 90% and 5% respectivelyof Thy1.1⁺ cells in mice given HCT, the CD8⁺ and CD4⁺ cells accountedfor 30% and 28% respectively of Thy1.2⁺ cells in mice without HCT.Almost all of the CD8⁺ and CD4⁺ T cells in mice given HCT wereCD62L^(lo) (FIG. 5B). The staining pattern indicates that few naïve orcentral memory cells were found in these tumors, almost all wereeffector memory cells, since the CD8⁺ and CD4⁺ cells were almost allCD44^(hi).

In contrast, the gated CD8⁺ and CD4⁺ cells from tumors in control micecontained discrete subsets of both CD62 L^(low) and CD62 L^(hi) cells.The CD62 L^(hi) cells accounted for 26% of CD8⁺ cells and 58% of CD4⁺cells (FIG. 5B). Staining of gated CD4⁺ tumor cells from control miceand those given HCT for CD4 versus CD25 showed that about 16% of CD4⁺cells were CD25⁺ in controls, and 32% were CD25⁺ in those given HCT atthe day 28 time point (FIG. 5C). At day 14, 22% of CD4⁺ cells wereCD25⁺. The results of additional staining for intracellular FoxP3⁺showed that the mean percentage of CD4⁺CD25⁺ FoxP3⁺ Treg cells amonggated CD4+ cells in the tumors of all 3 groups of mice varied from about15% to 25% (FIG. 5D).

The differences in the means were not statistically significant (p>0.05)as judged by the Student t test. Despite the similar percentages of Tregcells among total CD4+ T cells in the tumor cell infiltrate, there was amarked difference in the balance of CD8⁺CD62 L^(lo)CD44^(hi) effectormemory T cells versus Treg cells. Whereas, day 28 tumors from controlmice showed a mean ratio of about 5:1 CD8⁺ effector memory to Treg cellsin the infiltrate, the day 28 tumors from mice given HCT showed meanratio of about 50:1. The differences in ratios were statisticallysignificant (p<0.05). Thus, while HCT did not deplete Tregs at the tumorsite, the balance of tumor infiltrating cells was altered to favor CD8⁺effector memory T cells as compared to Treg cells.

Example 6 Tumor Vaccination becomes Effective when Combined with HCT andVaccine Induced Anti-Tumor Immunity is not Prevented by the Presence ofGrowing Tumors

FIG. 6A shows the experimental scheme used to determine the effect ofvaccination alone on survival of tumor-bearing mice. FIG. 6B shows thatthe survival of vaccinated, but not HCT treated, animals with 7-daytumors did not improve as compared to unvaccinated tumor-bearing animalsand all animals died by day 40. Moreover, when vaccinated nontumor-bearing animals were challenged with as few as 2.5×10⁴ CT26 cells16 and 50 days after vaccination (FIG. 6C), only 20% of mice survived100 days (FIG. 6D). Some degree of protection developed in animalsvaccinated given the tumor challenge 90 days after vaccination, asindicated by the observation that 50% of animals remained tumor free(p=0.03) (FIG. 6D). Next, to assess the potential effect of largertumors on the response to vaccination, we vaccinated mice with tumorsgrowing for 7 days and then waited 14 additional days before resectingthe growing tumors at day 21 (FIG. 6E). Bone marrow and splenocytes wereharvested from donors on day 110 and transferred into lethallyirradiated tumor-bearing hosts. All hosts survived with complete tumorregression for at least 100 days (FIG. 6F). Only 20% of hosts giventransplants from unvaccinated donors survived 100 days, and thedifference using vaccinated versus unvaccinated donors was significant(p<0.05) (FIG. 6F). Thus, HCT from vaccinated animals into syngeneictumor bearing hosts resulted in cure of tumors, indicating that growingtumors in donors do not prevent the development of potent anti-tumoreffector cells in adoptive hosts.

Example 7 Autologous HCT Enhances Tumor Immunity after Vaccination

A model of autolous HCT was studied as shown in FIG. 6G. In this schemea group of donors was vaccinated 7 days after live tumor cell injection,splenectomized 7 days later, conditioned with TBI immediately afterrecovery from surgery, and spleen cells were injected intravenouslywithin 6 hours after TBI. Bone marrow cells were not required for rescueof these myeloablated hosts, since mouse spleen cells contain bothimmune cells and HSCs. Donors that received the autologous transplantshad significantly improved survival as compared to those withouttransplants, and about 40% survived at least 100 days with completetumor regression (p<0.001) (FIG. 6H). Thus, large 14 day tumors wereeither cured or their growth significantly delayed after autologous HCT.

Example 8 Clinical Study to Assess the Safety and Feasibility ofAutologous Tumor Cell-TLR9 Agonist Vaccination Prior to AutologousHematopoietic and Immune Cell Rescue in Metastatic Colorectal Cancer

Objectives

Primary Objectives

-   -   To assess the feasibility of using an autologous tumor cell        vaccine in combination with standard chemotherapy followed by        investigational autologous hematopoietic and immune cell rescue        in terms of acceptable clinical toxicity.

Secondary Objectives

-   -   To assess the clinical and immunologic efficacy of this vaccine        and autologous transplant regimen by measuring the following:        -   Ex vivo assessment of immune response        -   Response        -   Time to progression (TTP)

Background

As the third most common cancer in incidence and second in mortality,colorectal cancer (CRC) significantly impacts the lives of manyAmericans (25). In 2008, it is estimated that 148,810 cases will bediagnosed and 49,960 patients will die from this disease. Approximately20% of patients present with metastatic disease at diagnosis. Theintroduction of more effective chemotherapy regimens and biologicallytargeted agents over the last few years has led to considerableimprovement in treatment options for metastatic CRC yet median survivalapproximates only 2 years. Resection of the primary tumor whenclinically indicated followed by combinations of oxaliplatin oririnotecan with intravenous or oral 5-FU, leucovorin, and bevacizumabfor first-line therapy of metastatic CRC is standard of care.

In 2004, Goldberg and colleagues established FOLFOX4 as the standard ofcare chemotherapy regimen in metastatic CRC when they demonstrated itssuperiority over two older regimens, IFL (bolus5-FU/leucovorin/irinotecan) and IROX (irinotecan/oxaliplatin), in termsof prolonging median overall survival (OS), progression-free survival(PFS), and increased response (26). FOLFOX4 increased median survivaltime to 19.5 months compared to 15 months and 17.4 months for IFL andIROX respectively (p=0.0001; HR 0.66, 95% CI 0.54-0.82). Time toprogression was also significantly increased to 8.7 months compared to6.9 and 6.5 months (p=0.0014). Furthermore FOLFOX-4 effected a 45%overall response rate compared to 31% (p=0.002) and 35% (p=0.03) for IFLand IROX respectively. It also induced significantly less associatedgrade ≧3 nausea, vomiting, diarrhea, febrile neutropenia, anddehydration than the other two regimens.

Hoping to improve this regimen further, capecitabine, an oral pro-drugof 5-FU, was introduced. It has significant advantages over infusional5-FU including ease of administration with its oral formulation, lack ofinfusion-related toxicities, and decreased duration of hospitalizationand clinic time. Multiple trials have pitted capecitabine-basedtherapies against infusional 5-FU regimens and have shown comparableefficacy (27-32). Overall toxicity profiles are also comparable betweenthe two regimens with the exception of less myelosuppression and morehand-foot syndrome with capecitabine compared to theinfusional-5-FU-based regimens. Thus, in clinical practice, CAPDX(capecitabine-oxaliplatin) is largely considered to be a comparableregimen to FOLFOX, with significantly more convenient administration.

The addition of targeted therapies that inhibit vascular endothelialgrowth factor (VEGF) and endothelial growth factor receptor (EGFR) tothe 5-FU/LV regimens have further increased survival. Bevacizumab, amonoclonal antibody against VEGF, was approved for metastatic CRC in2004 after the pivotal phase III trial, AVF2107g, showed a significantimprovement in OS from 15.6 months to 20.3 months (HR for death, 0.66,p<0.001) with the addition of bevacizumab to IFL (33). PFS and responsewere also significantly increased from 6.2 months to 10.6 months(p<0.001) and 34.8% to 44.8% (p=0.004) respectively. The first phase IIItrial to evaluate the combination of bevacizumab with oxaliplatin-basedchemotherapy (FOLFOX-4 or CAPDX), NO16966, demonstrated that theaddition of bevacizumab improved PFS by 1.4 months (9.4 vs. 8.0 months,HR 0.83, p=0.0023) but overall response rates were similar (34). MedianOS also increased from 19.9 months in the placebo group to 21.3 monthsin the bevacizumab arm but was not statistically significant (HR 0.89,p=0.077).

Cetuximab, a mouse/human chimeric monoclonal antibody to EGFR, has alsoshown promise in metastatic CRC (35, 36). The BOND trial, a multicenterrandomized phase II trial showed a significant doubling of response rateand a 2.6 month increase in PFS with the combination ofirinotecan-cetuximab over cetuximab alone in the second line setting,but no difference in median OS (35). These results led the FDA toapprove cetuximab in February 2004 for second-line treatment either as amonotherapy in those who cannot tolerate irinotecan or in combinationwith irinotecan in those who do not have a response to irinotecan alone.The CRYSTAL trial, a phase III multicenter randomized trial alsodemonstrated that cetuximab holds promise in the first line setting(37). In this trial (n=1217), the addition of cetuximab to FOLFIRIsignificantly increased response rate by 6% (46.9% vs. 38.7%, p=0.005)and PFS by 1.9 months (p=0.036). Trials evaluating the first line use ofcetuximab with oxaliplatin-based regimens appear promising and areongoing (38, 39).

When using cetuximab, the presence of K-RAS mutations must beconsidered. Activating mutations in the K-RAS gene are present in 40-45%of colorectal cancer patients (40). The presence of these mutationscorrelates with a worse outcome and a lack of response to cetuximab inpatients with advanced chemotherapy-refractory CRC (41, 42).

Even with these new agents and improved combinations, median survivalfor metastatic CRC patients remains less than 2 years with less than 5%surviving to 5 years (43). Furthermore, one can expect 20% of patientsto progress within 4-6 months (34). Better regimens and treatments aregreatly needed to impact this pervasive and fatal disease.

Preclinical Data:

As shown in Examples 1-7, HCT from in vivo-immunized syngeneic donorscan cure solid tumors in mice, most likely due to peripheral memory Tcell response against the tumor antigen. Specific timing and primingconditions are critical, while the effect appears to be independent fromallo-immune reactivity. By combining this vaccine system with autologoushematopoietic cell transplantation, complete response was achieved in60-100% of mice. The response percentage depended on tumor burden.

Rationale

In summary, preclinical data demonstrate that HCT from in vivo-immunizedsyngeneic donors can cure solid tumors, most likely due to peripheralmemory T cell response against the tumor antigen. Specific timing andpriming conditions are critical, while the effect appears to beindependent from allo-immune reactivity. By combining this vaccinesystem with autologous hematopoietic cell transplantation, completeresponse was achieved in 60-100%. The response percentage depended ontumor burden and sufficient time for priming. Given these data andresults from the discovery and the present invention, it is expectedthat the preclinical studies discovery can be translated to humans withmetastatic CRC using the patients own tumor cells, processing the tumorcells to increase antigenicity, and by combining this vaccine andautologous hematopoietic and immune cell rescue with standard of careresection and, when needed, chemotherapy.

The preclinical data suggest that by selecting appropriate patients(e.g., those with a reasonable amount of tumor burden) and providingsufficient time for immunologic response, one can have effectivetreatment and potentially a cure in metastatic colon cancer patientsusing immunotherapy in conjunction with standard chemotherapy. Thistranslational endeavor may significantly impact the prognosis ofmetastatic colon cancer patients, who currently face a median overallsurvival of about 2 years from diagnosis.

Tumor Processing

Based on prior reports and the pre-clinicial data of resected tumorspecimens prepared as cell suspensions, approximately 1 cc of resectedtumor yields 1×10⁷ viable tumor cells (49, 51-58). Sufficient tumor willbe collected to allow recovery of ≧5×10⁷ viable cells aftercryopreservation and thawing. Vaccines are prepared and formulated asdescribed in section 4.1.5 using validated methodologies.

CpG

The term CpG denotes regions of DNA where a cytosine nucleotide existsnext to a guanine nucleotide. A phosphate (p) separates the twonucleotides. Unmethylated CpG dinucleotides occur more frequently inbacteria and viral genomes than those of vertebrates. The human immunesystem has evolved to detect these immunostimulatory motifs as a markerof infection. Krieg and colleagues first identified that bacterial DNAand synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpGdinucleotides induced murine B cells to proliferate and secreteimmunoglobulins in vitro and in vivo (59).

The immune system utilizes germline-encoded receptors to detectinfection via recognition of conserved molecular patterns associatedwith microbial pathogens (60). One class of these receptors is theToll-like receptors (TLRs). Bacterial CpG DNA is the natural ligand forTLR-9. Today, synthetic CpG ODN's are an established method to studyinnate immunity. When TLR-9 recognizes this immunostimulatory DNAcontaining unmethylated CpG motifs, innate immune responses areactivated that subsequently amplify the adaptive-immune response (61).Furthermore, when given in the presence of an antigen, CpG ODNs cantrigger predominantly Th-1 type immune responses (60). Th 1 immuneactivation is desired as it involves activation of NK cells andcytotoxic T lymphocytes (CTLs) that can kill tumor cells, much like theylyse cells infected by viruses and bacteria (62). Numerous animal modelshave demonstrated that synthetic CpG is an effective adjuvant thatenhances both humoral and cellular immune responses in diverseindications, ranging from infectious disease to cancer and allergy, andto date, clinical testing has largely affirmed the potency and safety ofISS-adjuvanted vaccines (61).

Its therapeutic potential in human studies is further advanced by itsease of synthesis, relatively inexpensiveness, and minimal toxicity(63). To date, CpG has been studied as an adjuvant to treatment in avariety of human cancers including lymphoma and non-small cell lungcancer. Various lymphoma and viral vaccine studies incorporating CpG asan adjuvant including doses of 6 mg and greater have shown mimimaltoxicity (61, 64-67).

Correlative Studies Background

Interleukin-7 (IL-7) & Interleukin-15 (IL-15)

IL-7, a cytokine produced by the bone marrow and thymic stroma, is anon-redundant cytokine essential for thymopoiesis as well as T cellsurvival, proliferation, and cytotoxic function in the peripheralcirculation (76). Expanding on IL-7's known function of inducingproliferation of B cell progenitors in vitro and in vivo, Bolotin andcolleagues also demonstrated that administration of IL-7 significantlyenhanced immune recovery after T cell-depleted bone marrowtransplantation (BMT) in a murine model (77). In another study, the samegroup explored the relationship between IL-7 and lymphocyte recoveryafter BMT (78). Lymphopenic patients with severe combined immunedeficiency (SCID) or acute lymphoblastic leukemia (ALL) and controlpatients less than 1 year old had higher serum levels of IL-7 by ELISA.Levels rose in response to further lymphopenia in post-transplantperiod. These findings underscore IL-7 as a likely regulator of de novoproduction of T lymphocytes after BMT. The authors assert the increasedIL-7 levels observed in the lymphopenic patients were due to alteredconsumption as these patients likely have decreased numbers of IL-7receptor (R)-bearing cells with resultant abnormal clearance of IL-7 asopposed to direct upregulation of IL-7 production in response tolymphopenia. They further propose that the binding of the IL-7R is ahomeostatic mechanism that regulates circulating levels of IL-7. Furtherinvestigation into regulation of IL-7 is needed. It is unclear if IL-7gene expression by stromal cells is up-regulated by any externalstimuli, but it may be negatively regulated by TGF-β, IL-1, and IFN-γ(78, 79). The degree of IL-7 production and resultant serum levels arealso likely a function of the age-dependent decline in thymic function(78, 80).

IL-15 is an important cytokine in the proliferation and survival ofnaïve, effector, and memory T cells. While enhancing the homeostaticproliferation of naïve T cells that require IL-7 and TCR interaction,crucial components of its receptor are not expressed until after naïve Tcells begin homeostatic proliferation in response to IL-7 (81). Inaddition to promoting the maturation and survival of naïve cells, aconsiderable body of work suggests that IL-15's primary role isregulation of the CD8⁺ T cell compartment as it induces proliferation ofmemory cells with cytolytic function (81, 82).

Cytokines that signal through receptors containing the common γ-chain(γc) such as II-7 and IL-15 are crucial for T-cell development,survival, expansion, and activation (81). Furthermore, IL-7 and IL-15are vital to the homeostatic response to T cell depletion in the settingof hematopoietic cell transplantation (76, 78). IL-7 appears to be moreessential for survival of memory T cells while IL-15 promotesproliferation of those same cells (82). In order to assess IL-7- andIL-15-associated homeostatic expansion of T cells, serum levels of thesecytokines will be measured at serial times before and afterhematopoietic and immune cell rescue.

Proliferation Assays

The combination of plasma assays for IL-7 and IL-15 with proliferationassays such as Ki67-labeling in T lymphocytes and TCR rearrangementexcision circles (TREC) analysis will be utilized to assess T cellexpansion.

Ki-67

Ki-67 is a well established intracellular method for detection of T cellproliferation in peripheral blood and will be performed serially beforeand after hematopoietic and immune cell rescue (82, 83).

TCR Rearrangement Excision Circles

During thymocyte development, rearrangement of the T-cell-receptor (TCR)gene results in excision of circular DNA fragments from genomic DNA(84). Douek and colleagues developed an assay that estimates thymicoutput more accurately by measuring the numbers of these TREC (85).TRECs are unique to T cells and diluted out with each cellular division,making them useful markers of developmental proximity to the thymus, andthus, their concentrations in peripheral blood can be used to estimatethymic output (85, 86). This technique compares favorably to oldertechniques that used T cell surface molecules as markers for recentthymic emigration and likely underestimated thymic activity.

Two distinct mechanisms repopulate the total T cell pool afterhematopoietic cell transplantation (86). An early rise especially inCD8⁺ cells after engraftment is mediated by the rapid expansion oflimited subsets of pre-existing mature T cells. This occurs in the first100 days and then declines. Conversely, the numbers of TREC whichmeasure the production of new naïve T cells from hematopoietic stemcells or more committed progenitor cells continue to increase steadilyuntil 1 year post-transplant. Doeuk and colleagues' data stronglysuggest that it is residual thymic tissue that is the most likely sourcefor the new TREC-positive naïve T cells that reconstitute the immunesystem after transplantation (86). Thus, the measurement of numbers ofTREC in peripheral blood may serve as a clinical indicator of thepotential for recovery of damaged immune system (86) and the techniquewill be utilized in this study to quantify the proliferation of newlymphocytes.

In vitro Response to Recall Antigens

In vitro immune responses to recall antigens tetanus toxoid, herpessimplex virus, varicella virus, and cytomegalovirus will be performed(86) before and after vaccination as well as pre- and post-rescue.

Cell-Mediated Lympholysis (CML)

In most preclinical models of hematopoietic cell transplantation,eradication of tumor is mediated by CD8⁺ T cells using a directcytolytic pathway. Using a standard CML assay, CD8⁺ and CD4⁺ cytolyticfunction will be measured using the patient's purified blood T cellspre- and post-rescue.

Preclinical and early clinical work has shown that cytotoxic Tlymphocytes (CTLs) generated against autologous tumor induce substantialanti-tumor activity. In the current clinical protocol, anti-tumor immuneresponses will be tested serially in vitro before and after vaccinationand pre-and post-rescue. Purified total T cells or purified CD4⁺ andpurified CD8⁺ T cells (and their memory subsets) will be used asresponder cells (50×10³/well). Stimulator cells will be irradiated(5,000 cGy in vitro) single cell suspensions of tumor cells (50×10³) orpurified dendritic cells (86) pulsed with the tumor cell lysate obtainedby sonication and freeze/thaw. Measurements of responses will includeproliferation (³H-thymidine incorporation) and production of thecytokines IL-2, IFN-γ, TNF-α, and IL-10 assayed by intracellularstaining or in triplicate culture well supernatants by ELISA (86).

Autoimmunity Panel

A wide range of autoimmune reactions have been seen withimmunomodulatory agents such as IFN-α, IL-2, and anti-cytotoxicT-lymphocyte antigen 4 (CTLA-4) including thyroiditis, inflammatorybowel disease and enteritis, hepatitis, vitiligo, dermatitis, arthritis,vasculitis, hypophysitis, panhypopituitarism, and anti-phospholipidsyndrome (87-90). In turn, these autoimmune responses often appear to beassociated with antitumor responses (88, 90). Gogas and colleaguesdemonstrated that autoimmunity was an independent prognostic marker forimproved relapse-free survival and overall survival in patients withmelanoma who received adjuvant IFN-α-2β.⁶⁴ Autoimmunity was observedafter a median of 3 months and in some cases after more than a year fromIFN-α initiation (91). In the Gogas trial, serum was tested foranti-thyroid, antinuclear, anti-DNA, and anti-cardiolipin antibodies andpatients were examined for vitiligo. As one of the goals of ourinvestigational therapy is to induce an autoimmune response against thetumor, it is reasonable to assess for autoantibodies or clinicalmanifestations of autoimmunity. Serum will be assessed serially beforeand after hematopoietic and immune cell rescue for increased levels ofautoantibodies including antithyroid antibodies (anti-thyroglobulin,anti-microsomal antibodies), antinuclear antibodies, anti-doublestranded DNA antibodies, and anti-cardiolipin antibodies. Rheumatoidfactor, thyroid stimulating hormone, thyroxine, and triiodothyroninelevels will also be measured. Investigators will assess for symptoms ofother autoimmune disorders during the history that might not be capturedon these laboratories, and patients will be examined for the clinicalmanifestations such as vitiligo on a regular basis.

Participant Selection and Enrollment Procedures

Inclusion Criteria

-   -   Histologically confirmed Stage IV, T×N×M1 colon adenocarcinoma        with a surgically accessible primary or metastatic site.    -   Estimated survival of 6 months or greater    -   Primary may be in place    -   Age 18-70    -   Must have an ECOG performance status of 0 or 1    -   Must have adequate organ and marrow function. Specifically:        -   Absolute neutrophil count (ANC) >1500/μL        -   Platelet count ≧100×10⁹/L        -   Total bilirubin ≦2.0× the upper limit of normal (ULN)        -   Alkaline phosphatase, AST, and/or ALT <2.5× the ULN for            patients without evidence of liver metastases; <5× ULN for            patients with documented liver metastases        -   Serum creatinine <2.0 mg/dL        -   Hemoglobin >9 g/dL            -   Patients may be transfused or receive epoetin alfa to                maintain or exceed this level up to the hemoglobin level                recommended on the current label for epoetin alfa. There                is concern that hemoglobin levels greater than the level                recommended by the current labeling have been associated                with the potential increased risk of thrombotic events                and increased mortality. Also, a rapid increase in                hemoglobin may exacerbate hypertension (a concern in                patients with pre-existing hypertension and if                bevacizumab is administered).        -   Cardiac ejection fraction >40% by transthoracic echo or MUGA            scan within 12 wks of transplant        -   Adequate pulmonary function tests (PFTs) within 6 wks of            transplant            -   DLCO ≧60% predicted        -   Patients must be HIV negative    -   No prior therapy which would preclude the use of total body        irradiation    -   Pathology must be reviewed and diagnosis confirmed by Stanford        University Medical Center    -   Ability to understand and the willingness to sign a written        informed consent document.    -   Ability and capacity to comply with the study and follow-up        procedures.        Exclusion Criteria

Disease-Specific Exclusions

-   -   Radiotherapy within 28 days prior to the day of tumor resection        (Day 1).    -   No myelosuppressive chemotherapy within 28 days prior to the day        of tumor resection    -   History of brain metastases, regardless if treated.

Co-morbid diseases or intercurrent illness

-   -   Active infection or fever >38.5° C. within 3 days of starting        treatment    -   History of other malignancies within 5 years prior to Day 1        except for tumors with a negligible risk for metastasis or        death, such as adequately controlled basal cell carcinoma,        squamous-cell carcinoma of the skin, carcinoma in situ of the        cervix, early-stage bladder cancer, or low-grade endometrial        cancer    -   Malignancies that have undergone a putative surgical cure (i.e.,        localized prostate cancer post-prostatectomy) within 5 years        prior to Day 1 may be discussed with the Medical Monitor.    -   History or presence of autoimmune disorders requiring treatment    -   Any other medical conditions (including mental illness or        substance abuse) deemed by the clinician to be likely to        interfere with a patient's ability to provide informed consent,        cooperate, or participate in the study, or to interfere with the        interpretation of the results.    -   Inadequately controlled hypertension (defined as systolic blood        pressure >150 and/or diastolic blood pressure >100 mmHg on        antihypertensive medications)    -   Any prior history of hypertensive crisis or hypertensive        encephalopathy    -   New York Heart Association (NYHA) Grade II or greater congestive        heart failure (see Appendix A)    -   History of myocardial infarction or unstable angina within 6        months prior to study enrollment    -   History of stroke or transient ischemic attack within 6 months        prior to study enrollment    -   Significant vascular disease (e.g., aortic aneurysm, aortic        dissection)    -   Symptomatic peripheral vascular disease    -   Evidence of bleeding diathesis or coagulopathy that is not        intentionally pharmacologically-induced    -   Serious, non-healing wound, ulcer, or bone fracture    -   Proteinuria at screening as demonstrated by either:        -   Urine protein: creatinine (UPC) ratio ≧1.0 at screening OR        -   Urine dipstick for proteinuria ≧2+ (patients discovered to            have ≧2+ proteinuria on dipstick urinalysis at baseline            should undergo a 24 hour urine collection and must            demonstrate ≦1 g of protein in 24 hours to be eligible).

Radiation-specific exclusions

-   -   Prior radiation to >25% of the marrow

Pregnancy

-   -   Women who are pregnant or breast feeding, or women/men able to        conceive and unwilling to practice an effective method of birth        control.        -   Women of childbearing potential must have a negative urine            or serum pregnancy test within 7 days of study entry.    -   Nursing patients will be excluded        Treatment Plan

Investigational Agent Administration

We will undertake a pilot trial to assess the safety and feasibility oftreating adult metastatic colorectal cancer patients who have asurgically accessible sterile primary or metastatic site with anautologous tumor cell/TLR9 agonist vaccine followed by autologoushematopoietic and immune cell rescue.

Screening

The patients of the gastrointestinal medical oncology and surgicaloncology practices of physicians in a major Cancer Center will bescreened for the inclusion and exclusion criteria described in sections3.1 and 3.2. Basic eligibility requires adult (aged 18-70 years)metastatic colon cancer patients who have a life expectancy greater than6 months as well as a surgically accessible sterile primary ormetastatic site and an ECOG performance status of 0 or 1. The trial, itsgoals, risks and benefits will be extensively discussed with the patientand they will be given adequate time to review the written informedconsent and ask questions. A written informed consent must be signedprior to trial entry.

During the screening period, patients will have a complete history andphysical examination performed including demographics, vital signs,height, and weight.

Within 7 days of tumor resection, baseline labs including a CBC withdifferential, complete metabolic panel, CEA, CA 19-9, autoimmunitypanel, thyroid stimulating hormone, thyroxine, and triiodothyroninelevels will be obtained. Within 4 weeks of the baseline apheresis, allpatients will have a baseline CT scan of the chest/abdomen/pelvis,pulmonary function testing with spirometry and diffusing capacity,transthoracic echo or MUGA scan, and additional laboratory analysisincluding: hepatitis panel (HepBsAgA, HepB total core Ab, Hep total Ab,Hep C Ab, qualitative Hep C PCR), HIV-1 Ag, HIV 1 & 2 antibody, HIV PCR,HSV-1 & -2 Ab, HTLV-1 & -2 Ab, RPR, VZV Ab, CMV IgG & IgM, and baselineautoimmunity screen (ANA, anti-double stranded DNA, anti-microsomalantibodies, anti-thyroglobulin, anti-cardiolipin, rheumatoid factor).

Central Line Placement

Insertion of a 12 French Cook catheter will be done prior to thebaseline apheresis.

Baseline Apheresis

Prior to metastectomy and if needed, primary tumor resection, patientswill undergo a first apheresis to establish baseline immune markers.This apheresis will be performed in the blood bank in the standardmanner. The goal of this apheresis is twofold: (1) to obtain >10⁸ PBMCsand lymphocytes which will be frozen in aliquots of 1 to10 million, and(2) to collect 100 mL of autologous plasma for use in tumor vaccinepreparation and cryopreservation to avoid the use of allogeneic humanserum. The apheresis will take approximately 1 hour and approximately120 mL of blood will be removed which is roughly equivalent to 8tablespoons.

Resection

Within 1 week (preferably days) of the baseline apheresis, the patientwill be taken to the operating room for resection of tumor either in theform of metastatic disease or the primary when clinically indicated andusing standard surgical procedures.

Tumor Cell Processing and Vaccine Creation

For vaccination, patients must have cryopreserved autologous tumor cellsprepared as follows. The Surgical Pathology service will asepticallycollect freshly resected colon cancer tissue for vaccine preparation.Tumor specimens will be placed in cold (2-8° C.) medium consisting ofRPMI supplemented with 10% autologous plasma. In general, up to 5 g oftumor will be collected for vaccine preparation. Tumor samples will bemaintained cold and transferred to the Stanford Blood & MarrowTransplant (BMT) Laboratory for processing. Freshly resected tumors willbe dissociated under aseptic conditions into single-cell suspensions bymechanically mincing tumor into small pieces of approximately 5 mm³,followed by enzymatic digestion in Dulbecco's phosphate-buffered saline(DPBS) with an enzyme mixture (Liberase, Roche, Indianapolis, Ind.)containing collagenase types I and II, and deoxyribonuclease (Pulmozyme,Genentech, South San Francisco, Calif.). The digestion will be performedat room temperature with gentle agitation until dissociation iscomplete. The resulting cell suspension will be filtered through nylonmesh (Nytek; TETKO Inc, Briarcliff Manor, N.Y.). After washing with DPBS(Ca²⁺- and Mg²⁺-free), the cells will be resuspended in autologousplasma. Samples will be removed for cell count and viabilitydetermination, sterility assessment and endotoxin measurement. The tumorcell suspension will be concentrated to yield aliquots of 2×10⁷ cells in90% autologous plasma plus 10% dimethylsulfoxide (DMSO ProtidePharmaceuticals, Lake Zurich, Ill.) for cryopreservation. Vaccinealiquots will be frozen and stored in vapor phase liquid nitrogen at orbelow −155° C. until released for immunization and immunologic assay(see Section 5.2.1 for release testing criteria). Storage freezers arecontinuously monitored and equipped with remote alarms.

For vaccination, cryopreserved tumor cells will be thawed and washedtwice in DPBS. Ten to twenty million viable tumor cells in 1 ml will betransferred to the Stanford Blood Center and irradiated to a dose of 25Gy. The cells will be returned to the BMT Laboratory and resuspended in0.5-2 ml of DPBS containing 6 mg CpG to complete the vaccineformulation. Approximately 10% of the volume will be removed forlook-back sterility assessment of the vaccine. A dose of 1×10⁷ viablecells in up to 2 ml final volume will be loaded into a 2 cc syringe andreleased for vaccination as described below.

Vaccinations

All injections will be carried out at the GCRC or in the Stanford CancerCenter oncology clinic. Vaccinations will occur at weeks 1 and 2 aftersurgery (minimum of 7 days after surgery and 7 days between injections)and at a minimum of 7 days after autologous hematopoietic and immunecell rescue. Patients will be vaccinated subcutaneously at one site asper the vaccination schedule. Each dose of vaccine will consists of1×10⁷ autologous irradiated tumor cells and 6 mg CpG in DPBS.

Appropriate sites of vaccination include: the outer upper arm, abdomen,buttock, or outer thigh. The site of injection will be identified andshould be free of skin irritation. The area will be cleansed with analcohol or betadine swab. After the site dries, approximately 1-2 inches(2.5-5 cm) of skin will be held taut and the vaccine will be injected ata 45 degree angle using a 25-27 gauge ⅝″ needle and a 2 mL syringe.After needle withdrawal, pressure will be held over the site withsterile gauze until hemostasis is achieved. The site of injection willbe marked and the location will be recorded in the source documentation.

After each injection, the patient will be monitored for at least 1 hour.Vital signs will be taken 30 minutes after each injection.

Week 4 CT Scan of chest/abdomen/pelvis

-   -   If stable disease, patients will continue off chemotherapy.    -   If patients have evidence of progressive disease, capecitabine        monotherapy (1000 mg/m² twice daily for 14 days every 3 weeks)        or other minimally myelosuppresive but effective agent at will        be initiated. Patients will then continue with aphereses 2 and 3        at weeks 7 and 8 respectively.

Apheresis 2 (for Immune Cell Rescue)

At week 7, patients will undergo a second apheresis for a minimumcollection goal of >3×10⁸ unstimulated PBMCs/kg patient weight. PBMCswill be cryopreserved in autologous plasma with 10% DMSO at a dose of atleast 2×10⁸/kg for immune cell rescue. An additional 10 or more vialscontaining 1-10 million PBMCs will be cryopreserved for use in forfuture ex vivo experiments.

Apheresis 3 (for Hematopoietic Cell Rescue)

At 7-21 days after Apheresis 2, patients will receive G-CSF injectionssubcutaneously at a dose of 5-10 μg/kg/day for 4 days (goal Sundaythrough Thursdays). On day 4, the peripheral blood CD34+ cell count willbe checked. If the CD34+ count is <5/μL, patients will undergo apheresison day 4 followed by a fifth dose of G-CSF on day 5 and an additionalapheresis on day 5. The product of these collections will be batchedtogether. The combination of the two collections should be adequate forthe rescue. If the CD34+ count is >10, one collection will be done onthe afternoon of Day 5 (e.g., Thursdays). Using an Isolex 300 device,CD34+ progenitor cells will be isolated from the apheresis product asper manufacturer's instructions. These collections will becryopreserved.

CT Ccan of the Chest/Abdomen/Pelvis with Contrast will be Done at Week 8

-   -   If stable disease on or off chemotherapy, continue to        conditioning regimen of fludarabine and MI followed by        autologous hematopoietic and immune cell rescue.    -   If progressive disease, initiate or add other chemotherapeutic        agents [e.g., capecitabine and oxaliplatin (or        irinotecan)+/−bevacizumab] for 3 cycles, then reassess with        repeat CT scans at week 18. Appropriate chemotherapy regimens        will be determined by the investigators.    -   The risks and benefits of each chemotherapeutic or targeted        agent will be reviewed at the time of informed consent, at        treatment initiation, and whenever the patient has questions.

Week 18 Post-chemotherapy Re-staging CT Scans

If applicable, the patients will be assessed for response to thechemotherapy with a repeat contrast CT scan of the chest, abdomen, andpelvis during the rest week of cycle 3.

If re-staging CT scans of the chest, abdomen, and pelvis show completeresponse, partial response, or stable disease as defined by traditionalRECIST criteria (92), the patients will proceed to fTBI followed byautologous hematopoietic and immune cell rescue with re-infusion of theunstimulated and mobilized T cells gathered from aphereses 2 and 3 (seesection 4.1.8 & 4.1.9).

Patients, who have evidence of progressive disease on the week 18re-staging CT scan will be considered for second line regimens such ascetuximab and irinotecan or whatever regimen is deemed most appropriateby the investigators while adhering to standard of care practices. Iftheir performance status and organ function is inadequate for furtherchemotherapy, they will be discontinued from the trial. If second linetherapy induces stabilization of disease or partial response, thosepatients can then undergo fTBI followed by autologous hematopoietic andimmune cell rescue if they continue to meet the other eligibilitycriteria.

Autologous Hematopoietic and Immune Cell Rescue

Patients who have response on chemotherapy or stable disease on or offchemotherapy will then undergo an induction regimen of fludarabine 30mg/m² IV daily over 45 minutes for 3 days followed by fTBI. The dose ofthe fTBI will be determined according to the cohort the patient entersinto:

FTBI dose will be determined using a 3+3 dose escalation scheme with thefollowing dose levels:

-   -   Dose level #1: 400 cGy (administered as 200 cGy once daily for        two days)    -   Dose level #2: 600 cGy (administered as 200 cGy once daily for        three days)    -   Dose level #3: 800 cGy (administered as 200 cGy once daily for        four days)

For example, for dose level #1, the conditioning regimen schedule wouldbe:

Fludarabine Fludarabine Fludarabine fTBI fTBI + transplant ↓ ↓ ↓ ↓ ↓ ↓ ↓↓ ↓ ↓ D-4 D-3 D-2 D-1 D0 D-4 Fludarabine D-3 Fludarabine D-2 FludarabineD-1 fTBI D 0 fTBI followed by hematopoietic and immune cell transplant

At the higher dose levels, fludarabine would start 1 to 2 days earlierdepending on the number of days of fTBI.

See section 6.3.1 for details on dose escalation scheme.

The target of T cell depletion at the time of transplant is an ALC <0.1

Patients will be hydrated prior to, during, or after fTBI as clinicallyindicated.

Most patients will require anti-emetics or sedatives to decrease nauseaand vomiting. Furosemide may be utilized to maintain the patient'sweight at or near the admission baseline weight.

If HSV-1 or-2 positive on screening, acyclovir 400 mg orally twice dailywill be given starting the first day of fTBI until 1 year after the dayof rescue (Day 0).

The frozen hematopoietic and immune cells will be transported to theoutpatient Stanford Cancer Center BMT Infusion Treatment Area (ITA),thawed in a warm water bath, and infused through a central venouscatheter as rapidly as possible. Patients will be pre-medicated withhydrocortisone 100 mg IV and diphenhydramine 50 mg IV 30 minutes priorto cell infusion.

Post-Transplant Vaccine Boost

If patients have adequate coagulation parameters, i.e., platelets >50,000, INR <1.5, they will receive a vaccine boost at week 1 post-rescue.The boost will be a minimum of 7 days after transplant.

Post-Transplant Care

Patients will be prophylaxed against infection with the following:

-   -   If HSV-1 or-2 positive, acyclovir 400 mg p.o. bid starting on        first day of fTBI    -   When ANC <500, ciprofloxacin 500 mg orally daily will be        started.    -   Days +30-60: Bactrim 160 mg/800 mg p.o. bid, Saturday and Sunday        only.        -   No PCP prophylaxis will be given prior to D+30 as may delay            hematopoietic reconstitution    -   Serum CMV monitoring q week until Day +60

Neupogen will not be given initially as there is some evidence thatneupogen can induce a TH2 response in lymphocytes which may attenuateour CTL anti-tumor effect. Yet, if more than 2 of the first 6 patientsexperience neutropenic fever requiring hospital admission beyond 7 daysor prolonged neutropenia beyond 2 weeks, the investigators can eitherdecrease the fTBI dose according to the protocol or add neupogen 5mcg/kg sc daily starting day +6 until wbc >5000.

If admitted to the hospital, patients will be followed by the BoneMarrow and Transplantation (BMT) service following standard of practicefor post-autologous transplantation care.

If not admitted to the hospital, patients will be followed in the BMTDay Hospital for signs of infection. CBCs and BMT panels will beperformed a minimum or three times per week during the first 35 days.Weekly chest radiographs will be performed. A history and physicalexamination will occur at each visit.

Follow-Up after Hematopoietic Cell Reconstitution

After rescue, patients will be evaluated every month with physical examsincluding vital signs, basic laboratories, and CEA/CA 19-9 levels for 3months then every 2-4 months as clinically indicated. Immune monitoringwill be performed as described in section 4.1.15.3. CT scans of thechest/abdomen/pelvis will be done monthly for 2 months then every 2months to evaluate for progression. Patients who withdraw from treatmentdue to progressive disease will be seen within four weeks of thedetermination of progressive disease for a final study visit. Patientswho withdraw due to intolerance of treatment should be followed weeklyuntil all toxicities have reverted to Grade ≦2 or have stabilized in theopinion of the Investigator, at which point they will undergo the finalvisit. All patients who withdraw for any reason other than progressivedisease will be seen within 4 weeks of withdrawal for a final visit.

It is anticipated that several months will be required for the CTLs toexhibit antitumor activity. Patients with “symptomatic” diseaseprogression after rescue will be treated with best available systemictherapy. But when clinically appropriate, chemotherapy will be held forat least 3 months post-transplant to allow for the CTLs to proliferateand act.

Monthly for 3 Months Post-Rescue then Every 2-4 Months as ClinicallyIndicated

-   -   Physical exam including vital signs    -   Laboratories: CBC with differential, complete metabolic panel,        and if appropriate CEA and CA 19-9    -   Contrast CT chest/abdomen/pelvis if creatinine within normal        limits in month 1 and 2 then every 2 months

Final Visit

At the Final Visit, the following procedures will be performed:

-   -   Elicitation of adverse events and toxicity grading according to        the NCI CTC    -   Recording of concomitant medications    -   Complete physical exam including vital signs and ECOG        performance status    -   Laboratories:        -   CBC with differential and platelet count        -   Comprehensive metabolic panel        -   CEA and CA 19-9 if appropriate

Immune Monitoring Protocol

Daily Start Post- Week Test Screen A1 A2 A3 Cond. D0 rescue +2 +1 mo +3mo +6 mo +12 mo +18 mo Term. CBC with X X X X X X X X X X X XDifferential until ANC > 500 Absolute # of X  X* X X X CD3+/CD4+/CD8+^(a) In vitro resp. to X X X X X recall antigens^(b) IL-7, IL-15^(c)X X X X X X X X X % Proliferating X X  X* X X X T cells^(d) CEA, CA19-9^(e) X X X X X X X X In vitro X X  X* X X X antitumor immuneresp.^(f) Auto-immunity X X X X panel^(g) ^(a)CD3+/CD4+/CD8+: absolutenumbers/percentages including naive (CD62L⁺CCR7⁺CD45RA⁺), central memory(CD62L⁺CCR7⁺CD45RO⁺), and effector memory (CD62L⁻CCR7⁻CD45RO⁺) subsets.^(b)In vitro responses to recall antigens: tetanus toxoid, HSV,Varicella, CMV ^(c)Serum IL-7 and IL-15: assessment ofcytokine-associated homeostatic expansion of T cells ^(d)% ProliferatingT cells: Ki-67, TREC ^(e)CEA, CA 19-9: q month × 3 after transplant thenq2-4 months as clinically indicated and only if baseline level was high.^(f)Anti-tumor immune responses will assessed in vitro: purified total Tcells or purified CD4⁺ and purified CD8⁺ T cells (and their memorysubsets) will be used as responder cells (50 × 10³/well). Stimulatorcells will be irradiated (5,000 cGy in vitro) single cell suspensions oftumor cells (50 × 10³) or purified dendritic cells pulsed with the tumorcell lysate obtained by sonication and freeze/thaw. Measurements ofresponses include proliferation (³H-thymidine incorporation) andproduction of the cytokines IL-2, IFN-γ, TNF-α, and IL-10 assayed byintracellular staining or in triplicate culture well supernatants byELISA. ^(g)Autoimmunity panel: ANA, anti-ds-DNA antibodies, anti-thyroidantibodies, anti-microsomal antibodies, anti-thyroglobulin antibodies,anti-cardiolipin antibodies, rheumatoid factor, thyroid stimulatinghormone, thyroxine, and triiodothyronine levels *if ALC > 1500 Key: A:Apheresis; Screen: Screening; D: Day; Flud: Fludarabine; D0: Day ofhematopoietic and immune cell rescue; Mo: month

Screening

-   -   CBC with differential (ALC, ANC, platelets)    -   CEA, CA 19-9    -   Autoimmunity panel

Apheresis 1

-   -   CBC with differential (ALC, ANC, platelets)    -   CEA, CA 19-9 if applicable    -   T cell subsets    -   Assessment of in vitro response to recall antigens    -   IL-7, IL-15    -   Proliferating T cell indices: Ki-67, TREC    -   Assessment of in vitro antitumor immune response

Apheresis 2

-   -   CBC with differential (ALC, ANC, platelets)    -   Assessment of in vitro response to recall antigens    -   IL-7, IL-15    -   Proliferating T cell indices: Ki-67, TREC    -   Assessment of in vitro antitumor immune response

Apheresis 3

-   -   CBC with differential (ALC, ANC, platelets)

Start of conditioning: Day 1 of fludarabine

-   -   CBC with differential (ALC, ANC, platelets)    -   CEA, CA 19-9 if applicable    -   IL-7, IL-15

Day of autologous hematopoietic and immune cell rescue (Day 0)

-   -   CBC with differential (ALC, ANC, platelets)    -   IL-7, IL-15

Daily after rescue until ALC >500, ANC>500, platelets>20,000

-   -   CBC with differential (ALC, ANC, platelets)

Week +2 post-rescue

-   -   IL-7, IL-15

Month +1

-   -   CBC with differential (ALC, ANC, platelets)    -   T cell subsets (if ALC >1500)    -   CEA, CA 19-9 if clinically indicated    -   IL-7, IL-15    -   Proliferating T cell indices: Ki-67, TREC    -   Assessment of in vitro antitumor immune response

Month +3

-   -   CBC with differential (ALC, ANC, platelets)    -   CEA, CA 19-9 if applicable    -   T cell subsets    -   Assessment of in vitro response to recall antigens    -   IL 7, IL-15    -   Proliferating T cell indices: Ki-67, TREC    -   Assessment of in vitro antitumor immune response

Month +6

-   -   CBC with differential (ALC, ANC, platelets)    -   CEA, CA 19-9 if applicable    -   T cell subsets    -   Assessment of in vitro response to recall antigens    -   IL-7, IL-15    -   Proliferating T cell indices: Ki-67, TREC    -   Assessment of in vitro antitumor immune response    -   Autoimmunity panel

Month +12

-   -   CBC with differential (ALC, ANC, platelets)    -   CEA, CA 19-9 if applicable    -   T cell subsets    -   Assessment of in vitro response to recall antigens    -   IL-7, IL-15    -   Proliferating T cell indices: Ki-67, TREC    -   Assessment of in vitro antitumor immune response    -   Autoimmunity panel

Month +18

-   -   Autoimmunity panel

Study termination

-   -   CBC with differential (ALC, ANC, platelets)    -   CEA, CA 19-9 if applicable

As needed based on symptoms (Per the discretion of the investigator)

-   -   Imaging    -   Laboratory assessment

General Concomitant Medication and Supportive Care Guidelines

Supportive Medications

Nausea, vomiting, or both may be controlled with antiemetic therapy.

Mild to moderate allergic or hypersensitivity reactions can be treatedwith antihistamines such as diphenhydramine.

Unacceptable Concomitant Medications

The following medications may not be administered to study patientsduring the treatment period of the trial:

-   -   Pentastatin will be avoided during fludarabine administration        given the high incidence of pulmonary toxicity when given        concomitantly    -   Chemotherapy, biological therapy or radiotherapy other than        specified in the protocol.    -   Any investigational treatments

All other medical conditions should be treated according to currentstandards of care at the discretion of the Investigator. All concomitantmedications and therapies must be recorded on the appropriate CRF (CaseReport Forms), with indication, dose, route, frequency and date ofadministration. In general, concomitant medication should be stabilizedbefore screening and should remain constant during the course of thestudy, whenever possible. Any change must be documented on the CRF.

Duration of Active Therapy

From baseline apheresis to the vaccine boost 7 days after transplant,the various therapies (e.g. resection, vaccinations, chemotherapy, fTBI,autologous hematopoietic and immune cell rescue) will take approximately11 weeks if no chemotherapy is required to 28 or more weeks ifchemotherapy is required.

Duration of Follow Up

All patients, including those who discontinue protocol therapy early,will be followed clinically for 3 years after transplant for responseand progression; and if necessary after 3 years for survivalinformation.

Please refer to 4.1.15 for follow-up procedures on patients whocompleted all study treatments. In brief, they will be followed withmonthly visits x3 then every 2-4 months for 3 years.

For those patients that discontinued the protocol early, follow-up willbe every 2-4 months after their final study visit for updates oninitiation of subsequent-line therapy and overall survival . until thefinal analysis. Patients discontinuing the study due to a drug-relatedadverse event must be followed for four weeks or until resolution ofstabilization of the event.

Criteria for Removal from Study

Patients will be removed from the study if:

-   -   Progressive disease compromises organ function or performance        status making them ineligible for total body radiation or        autologous hematopoietic and immune cell rescue    -   Patient withdraws consent    -   Lost to follow-up    -   Death

A genuine effort must be made to determine the reason(s) why a patientfails to return for the necessary visits or is discontinued from thetrial. It will be documented whether or not each patient completed theclinical study. If for any patient, study treatment or observations werediscontinued the reason will be recorded on the appropriate case reportform.

Alternatives

Alternatives to this Study Include:

-   -   Standard of care treatment with systemic chemotherapy and        resection or radiation of specific tumor sites as clinically        indicated    -   Another clinical trial    -   Best supportive care

Planned Procedures for Protecting Against and Minimizing all PotentialRisks

Acquisition of tumor specimens for preparation of vaccine will beperformed by oncologic surgeons. Preferred sites will be intraperitonealmetastases, liver metastases, pulmonary disease, or in rare cases, theextraluminal portion of a bulky primary.

The patients will be closely monitored throughout the trial withfrequent physician visits and AE monitoring.

Investigational Agent and Procedure Information

Aphereses

Apheresis is not a routine part of colon cancer management and is thusconsidered an investigational component. This procedure will enable usto collect the unstimulated immunized cytotoxic T lymphocytes (CTLs)(apheresis 2), and the G-CSF stimulated hematopoietic progenitor cells(apheresis 3) as well as perform immune monitoring (apheresis 1, 2, &3). Cells from aphereses 2 and 3 will be transplanted back into thelymphodepleted patient with the autologous hematopoietic and immune cellrescue. The re-infused immunized CTLs will undergo homeostatic expansionwhich will ideally induce regression in residual tumor. The re-infusedhematopoietic progenitor cells will aide in cell recovery after thefractionated TBI, reducing the duration of cytopenia.

Risks

Complications are rare. Life-threatening complications are extremelyrare.

Potential risks and/or discomforts of apheresis include:

-   -   Nervousness, light-headedness, fainting    -   Infection    -   Bruising    -   Blood loss    -   Air embolism

Potential problems with the anticoagulant(s) used during the apheresisprocess include:

-   -   Muscle cramping    -   Numbness, tingling sensations    -   Chills    -   Feelings of anxiety    -   Nausea, vomiting    -   Bleeding

Supportive Care

Adverse effects will be treated symptomatically as clinically indicated.

Vaccine

Tumor Cell Processing

Vaccine preparation requires extensive handling of resected tumor togenerate single cell suspensions. In addition, the cells are combinedwith recombinant enzymes and CpG during processing. Release testing ofthe product to ensure its safety for injection will be according to thefollowing schedule.

Assay Test sample Method Release criteria Sterility Resected tumorTryptic Soy Broth No growth suspension Fluid Thioglycolate MediumSterility Biosafety cabinet Blood agar No growth settle plates EndotoxinResected tumor LAL Kinetic <5 EU/ml suspension Chromagenic Assay Cellcount Pre-freeze Manual >5 × 10⁷ cells (hemocytometer) Cell countPost-thaw Manual >1 × 10⁷ cells (hemocytometer) Cell viability Post-thawTrypan blue >50% viable exclusion Sterility Final formulation TrypticSoy Broth No growth Fluid Thioglycolate Medium

Sterility testing will use the BioMerieux BactAlert system with bothFluid Thioglycollate Medium and Tryptic Soy Broth. This system has beenvalidated within the Clinical Microbiology Laboratory at StanfordHospital and Clinics and has been shown to be at least as sensitive tomicrobial contaminants as standard USP compliant assays. Vaccinesshowing evidence of microbial contamination either by detectable growthin sterility cultures or endotoxin levels above the stated limit willnot be released for injection.

Vaccines showing evidence of microbial contamination either bydetectable growth in sterility cultures or endotoxin levels above thestated limit will not be released for injection.

CpG

Agent Accountability

The CpG will be kept in a secured location within the Blood and MarrowTransplantation Lab where only the investigators will have access to it.

Procedure

Injections will be administered by a person designated by theinvestigator (e.g., study nurses, physicians) in response to writtenorders from the investigator.

Risks

The most frequent adverse events seen with CpG agents have been thoserelated to the expected immunomodulatory pharmacologic effects. Theseinclude local injection reactions, systemic flu-like symptoms, andhematologic changes.

-   -   Local injection site reactions: pain, erythema, edema,        inflammation    -   Flu-like symptoms: fever, myalgia, arthralgia, fatigue,        headache, rigors, and/or musculoskeletal pain    -   Neutropenia and neutropenic fever    -   Thrombocytopenia    -   Infrequent hypersensitivity reactions    -   Rare possibility of autoimmune disorders: e.g. autoimmune        thyroiditis, Sjogren's syndrome    -   Potential for seroconversion for anti-single stranded and        double-stranded DNA antibodies, ANA, rheumatoid factor (can be        asymptomatic)    -   Sepsis and Sepsis-related adverse events    -   Rare cardiac ischemia, cardiac arrhythmia (most had a cardiac        history or recognized risk factors)

Supportive Care

-   -   For local injection site reactions:        -   Treat symptomatically with hot or cold compresses at the            site, acetaminophen for pain or inflammation, antihistamines            for pruritis.        -   Options for future injections: splitting injection into more            than one site, rotating the site of injection, avoiding            injection into a site with an existing local reaction, using            small gauge needles.    -   Symptomatic treatment to alleviate flu-like symptoms, e.g.        acetaminophen    -   Because of small risk of hypersensitivity reaction, patients        will be observed in the Oncology or BMT infusion center for 60        minutes after each injection.    -   Patients with significant cardiac disease or EF <40% will be        excluded from the study    -   Patients will be monitored closely for neutropenia, NF, and        sepsis

Conditioning Regimen

Fludarabine

Pharmacology

Fludarabine monophosphate is a purine analogue used extensively in thetreatment of lymphoid malignancies, particularly CLL and low grade NHL.Fludarabine is phosphorylated intracellularly in several steps to itsactive form 2-fluoroadenosine arabinoside triphosphate (93). Althoughfludarabine functions primarily as an antimetabolite it does affect bothdividing and non-dividing cells. Fludarabine may function as aradiosensitizer (94), which may enhance the immunosuppressive propertiesof our regimen but also the toxicity.

Procedure

Total doses of 90 mg/m² to 240 mg/m2 have been used as part ofminitransplant regimens with well-characterized and acceptable toxicity(95-97). In this trial, patients will be given 30 mg/m² IV over 45minutes in the outpatient BMT infusion center for 3 days prior to thestart of fTBI.

Toxicities

At the doses used in this study the main side effects will beimmunosuppression and myelosuppression.

Other adverse reactions that occur in >10% of cases include:

-   -   Cardiovascular: Edema    -   Central nervous system: Fever (60-70%), fatigue, pain, chills    -   Dermatologic: rash    -   Gastrointestinal: mild nausea/vomiting, anorexia, diarrhea,        gastrointestinal bleeding (3-13%)    -   Genitourinary: urinary tract infection    -   Hematologic: myelosuppression (nadir 10-14 days; recovery 5-7        weeks), anemia, neutropenia (grade 4: 59%; nadir: ˜13 days),        thrombocytopenia (50-55%; nadir: ˜16 days)    -   Neuromuscular & skeletal: generalized weakness, myalgia,        paresthesia    -   Ocular: visual disturbance; rare blindness    -   Respiratory: cough, pneumonia, dyspnea, upper respiratory        infection    -   Infection    -   Diaphoresis    -   Rare: autoimmune hemolytic anemia

Supportive Care

Anti-emetics, blood and platelet transfusions will be given asclinically indicated.

Other toxicities will be managed symptomatically as they arise.

No concurrent pentastatin will be administered given the high incidenceof fatal pulmonary toxicity when given in combination with fludarabine.

Fractionated Total Body Irradiation

fTBI is not a normai part of clinical management for colon cancer.However, its use as part of a hematopoietic cell preparatory regimen islongstanding and we will follow our institution's well-establishedguidelines.

Procedure

TBI will be given in 200 cGy fractions daily using up to a 15 mV linearaccelerator at a rate of <15 cGy/minute. Dosimetry calculations will beperformed by the radiation physicist. Patient positioning and treatmentplanning procedures will be performed according to institutionalstandard practice.

fTBI will be initiated the day after the last dose of fludarabine whichdepending on the dose cohort will either be day -1 if in the 400 cGycohort, day -2 if in the 600 cGy cohort or day -3 if in the 800 cGycohort.

Supportive Care

Beginning the day prior to irradiation, patients may receive intravenousfluids as clinically indicated on an outpatient basis. Most patientswill require anti-emetics and/or sedatives to decrease nausea andvomiting. Furosemide will be utilized to maintain the patients' weightat or near the admission baseline weight.

Toxicities Associated with FTBI Include:

-   -   Nausea and vomiting    -   Temporary alopecia    -   Cytopenias with resultant risk of life-threatening infections        and/or hemorrhage        -   Risk lasts until hematopoietic reconstitution    -   Parotid swelling with elevation of serum amylase (rare)    -   Radiation pneumonitis    -   Diarrhea    -   Oral mucositis    -   Probable permanent sterility in all patients    -   Long term survivors may develop cataracts; some may require        corrective surgery and the use of soft lenses    -   Rare cases of second malignancies    -   Skin irritation

Supportive Care

Diuretics, anti-pyretics and other agents will be given to manage thetoxicities as clinically indicated.

Toxicities During the Infusion should be Minimal

-   -   Volume overload    -   Fever, chills    -   DMSO odor: DMSO will be infused along with the cells. It is        excreted through the lungs. Its characteristic odor may be        noticeable for 2-3 days after the infusion.

Risks After the Rescue

Infection during the period of cytopenia. The period of cytopenia isexpected to be short as this is not a fully myeloablative conditioningregimen. Even without re-infusion of cells, period of cytopenia is notexpected to be more than 10 days.

Due to the nature of autologous hematopoietic cell transplantation,hospitalizations for febrile neutropenia are common and most ofteneasily managed. There are no baseline data with which to estimate therisk of hospitalization with the above dose levels though they areconsiderably lower than that which is routinely used in myeloablativetransplantation regimens for hematolymphoid malignancies.

Dosing Delays/Dose Modifications

Vaccine Dose

Potential AE's

If greater than grade 1 skin reaction occurs at the injection site,subsequent injections should be given in a different site or adjacent tothe first injection site.

Patients who experience toxicity according to that described in Table6-1 may have their doses held or discontinued. If any grade 3 injectionsite reaction or systemic reaction occurs as characterized byhypotension, anaphylaxis, laryngeal edema, or hospitalization, nofurther vaccinations will be given.

TABLE 6-1 Autologous tumor cell-CpG 7909 Vaccine Dose Modifications CTCGrade Toxicity During Any Treatment Cycle Grade 3 injection site Nosubsequent vaccinations reaction or anaphylaxis Other Grade 3 or 4^(a)Omit dose until toxicity ≦ Grade 2, then resume dose

Fludarabine

No dose modifications possible.

ffBI

Dose Escalation Rules

No dose modifications possible at each level but the dosing will beaccording to a 3+3 dose escalation theme:

-   -   The first three patients will be accrued to cohort #1        sequentially with no more than one accrued every 4 weeks.        -   If none of the 3 patients require hospital admission for >7            days for any reason or develop grade 4 non-hematologic            toxicity, then the next three patients will accrue to dose            level #2.        -   If one of the 3 patients has a hospitalization >7 days or            grade 4 non-hematologic toxicity, then 3 more patients will            be accrued sequentially to dose level #1. If 2 of 6 patients            require hospitalization for >7 days or have grade 4            non-hematologic toxicity, accrual will be discontinued and            amendments to the study considered. If the subsequent three            patients do not require hospitalization for >7 days or            develop grade 4 non-hematologic toxicity, then the next            three patients will escalate to dose level #2.        -   The above rules also apply to escalation from dose level #2            to #3.    -   If two or more of the six patients accrued to dose level #2 or        #3 require hospitalization for >7 days or develop grade 4        non-hematologic toxicity, then accrual will continue at the        lower dose level (i.e. 800 cGy decreased to 600 cGy or 600 cGy        decreased to 400 cGy) until 9 patients have been treated.        Autologous Hematopoietic and Immune Cell Transplant

No dose modifications possible.

Adverse Events and Reporting Procedures

Definitions of Adverse Events

Adverse Events

An Adverse Event (AE) is the development of an undesirable medicalcondition or the deterioration of a pre-existing medical conditionfollowing or during exposure to a pharmaceutical product, whether or notconsidered causally related to the product. An undesirable medicalcondition can be symptoms (e.g., nausea, chest pain), signs (e.g.,tachycardia, enlarged liver) or the abnormal results of an investigation(e.g., laboratory findings, ECG). In clinical studies, an AE can includean undesirable medical condition occurring at any time, including run-inor washout periods, even if no study treatment has been administered.

Any detrimental change in a patient's condition subsequent to thementering the study and during the follow-up period should be consideredan AE. When there is a deterioration in the condition for which thestudy treatment is being used, there may be uncertainty as to whetherthis is lack of efficacy or an AE. In such cases, unless the reportingphysician considers that study treatment contributed to thedeterioration or local regulations state to the contrary, thedeterioration should be considered a lack of efficacy. Signs andsymptoms of disease progression are therefore not considered AEs.

The development of a new cancer should be regarded as an AE. New cancersare those that are not the primary reason for administration of studytreatment and have been identified after inclusion of the patient intothe clinical study.

Serious Adverse Event

A Serious Adverse Event (SAE) is an AE occurring during any study phase(i.e., run-in, treatment, washout, follow-up), and at any dose of theinvestigational product, comparator or placebo, that fulfills one ormore of the following criteria:

-   -   Results in death    -   Is immediately life-threatening    -   Requires inpatient hospitalization or prolongation of existing        hospitalization    -   Results in persistent or significant disability or incapacity    -   Is a congenital abnormality or birth defect    -   Is an important medical event that may jeopardize the patient or        may require medical intervention to prevent one of the outcomes        listed above.

Any event or hospitalization that is unequivocally due to progression ofdisease, as determined by the investigator, must not be reported as anSAE. The causality of SAEs (their relationship to all study treatment)will be assessed by the Investigator.

Reporting of Adverse Events

Adverse events will be recorded at each visit, which is the first day ofeach cycle. If an adverse event occurs mid-cycle requiring medicalattention, this will be recorded as well. The variables to be recordedfor each adverse event include, but are not limited to, onset,resolution, intensity, action taken, outcome, causality rating, andwhether it constitutes an SAE or not.

The intensity of the adverse event should be captured using CTCAEcriteria, version 3.0, when possible.

Pregnancy should be excluded before enrollment. Should a pregnancyoccur, it must be reported in accordance with the procedures describedin Section 8.2. Pregnancy in itself is not regarded as an AE unlessthere is a suspicion that an investigational product may have interferedwith the effectiveness of a contraceptive medication.

All non-serious adverse events will be reported to the FDA, IRB, andSRC.

If needed for progressive disease during this trial, chemotherapy willnot be considered investigational. Unless unusual for the particularchemotherapy, Grade 3 or 4 AEs attributed to standard chemotherapy (whenapplicable) will not be considered reportable AEs.

Reporting of Serious Adverse Events

Investigators and other site personnel must inform the FDA, via aMedWatch form, of any serious or unexpected adverse events that occur inaccordance with the reporting obligations of 21 CFR 312.32, and willconcurrently forward all such reports to the FDA, IRB, and SRC. It isthe responsibility of the investigator to compile all necessaryinformation and ensure that the FDA receives a report according to theFDA reporting requirement timelines and to ensure that these reports arealso submitted to the IRB and SRC at the same time.

A cover page should accompany the MedWatch form indicating thefollowing:

-   -   Investigator Sponsored Study (ISS)    -   The investigator IND number assigned by the FDA    -   The investigator's name and address    -   The trial name/title

The SAE report will designate the causality of events in relation to allstudy medications and if the SAE is related to disease progression, asdetermined by the principal investigator.

If a non-serious AE becomes serious, this and other relevant follow-upinformation will also be provided to the FDA, IRB, and SRC.

All SAEs will be documented. The investigator is responsible forinforming the IRB and/or the Regulatory Authority of the SAE as perlocal requirements.

All serious adverse events will be reported within 24 hours of firstknowledge of the event's occurrence, the “Serious Adverse Event Report”must also be sent whether or not complete information is available atthe time. If complete information is unavailable the Investigator mustprovide follow-up information to the FDA, IRB, and SRC as soon as it isknown. In particular, the Investigator must inform these groups by phoneand fax within 24 hours of occurrence of immediately life-threateningSAEs or SAEs with fatal outcome.

Correlative Studies

Laboratory Correlative Studies

All patient samples required for the studies detailed above will becollected and in both the overall study calendar and the immunemonitoring study calendar. The BMT lab will handle the preservation andshipping of specimens.

Study Calender

Day Cond. of (Flud. + HICR Day Q 2-4 Mo. Final Pre- Wk 1 Wk 2 Wk 4 Wk 7Wk 8 fTBI) (Day of Wk Mo Mo Mo after Study study Sx. V1 V2 CT A2 A31^(st) day 0) Boost +2 +1 +2 +3 Month +3 visit Informed consent XDemographics X History X Concurrent meds^(a) X X X X Physical exam^(b) XX X X X X X X Vital signs^(c) X X X X X X X X X Height X Weight^(d) X XX Performance X X X status^(e) CBC with Diff, X X X X X X X X X X PltsCMP X X X X X X X PT, INR X Infectious disease X screen^(g) AutoimmunityX X panel^(h) Serum/urine X X pregnancy test^(i) Central line Xplacement Apheresis^(j) X X X Tumor resection X Tumor X X X X X X Xmeasurements^(k) Radiologic eval: X X X X X X CT scan C/A/P or MRI^(l)TTE or MUGA, X PFTs^(m) Standard X X X chemotherapy as needed^(o)Adverse event X X X X X X X X X X X X X evaluation^(p) Fludarabine^(q) XfTBI^(r) X Auto HICR^(s) X Acyclovir ppx^(t) X Bacterial ppx^(u) X CMVmonitoring^(v) X Immune X X X X X X X  X^(l) X monitoring^(w) Othertests, as appropriate^(x) Survival X assessment^(y) Key: CT: computedtomography, C/A/P: chest/abdomen/pelvis, CMP: complete metabolic panel;Cond: conditioning; D: day, Flud.: fludarabine; fTBI: fractionated totalbody irradiation, HICR: hematopoietic and immune cell rescue, Mo.:month, MRI: magnetic resonance imaging, ppx: prophylaxis, Q: every, Sx:surgery/resection of tumor; Wk: week ^(a)Concurrent medications will beassessed by an investigator or ITA nurse at each visit or procedure^(b)Complete physical examination including neurologic assessment willbe done at screening, 1-2 days prior to resection and fTBI, day ofautologous HSCT, and at termination visit. Otherwise, brief disease- andadverse event-focused history and focused physical exam will be done.^(c)Blood pressure, heart rate, and temperature at screening, day ofresection/autologous HSCT. Blood pressure and heart rate will beassessed before and 30 minutes after vaccinations. ^(d)Weight will beassessed at screening, day of resection/vaccinations/autologous HSCT,and at monthly clinic visits. ^(e)Performance status will be assessedusing the criteria of the Eastern Cooperative Oncology Group (ECOG).^(f)CEA and CA 19-9 will only continue to be checked if high on initialscreening ^(g)Infectious disease screen: hepatitis panel (HepBsAgA, HepBtotal core Ab, Hep total Ab, Hep C Ab, qualitative Hep C PCR), HIV-1 Ag,HIV 1 & 2 antibody, HIV PCR, HSV 1 & 2 Ab, HTLV-1 & 2 Ab, RPR, VZV Ab,CMV IgG & IgM ^(h)Autoimmunity panel: ANA, anti-ds-DNA antibodies,anti-thyroid antibodies, anti-microsomal antibodies, anti-thyroglobulinantibodies, anti-cardiolipin antibodies, rheumatoid factor, thyroidstimulating hormone, thyroxine, and triiodothyronine levels ^(i)Forwomen of childbearing potential only. For all other women, documentrationale in their medical history that confirms why patient is not ofchildbearing potential. ^(j)Aphereses: Pre-study: apheresis within 7days prior to surgery for baseline immune markers Wk 7: apheresis forunstimulated cytotoxic killer lymphocytes (CTLs) Wk 8: G-CSF stimulatedapheresis for hematopoietic progenitor cells (HPCs) ^(k)Tumormeasurements: clinically for palpable lesions and radiographically usingRECIST criteria; Also to be done at week 18 if still on chemotherapy.^(l)Tumor assessment for all lesions will be evaluated according toRECIST criteria. ^(m)Within 4 weeks of the baseline apheresis, pulmonaryfunction testing with spirometry and diffusing capacity, andtransthoracic echo or MUGA scan will be performed to ensure adequatecardiac and pulmonary reserve. ^(n)Vaccinations will be performedsubcutaneously at a minimum of seven days after tumor resection andafter hematopoietic and immune cell rescue. The second vaccination willbe given a minimum of 7 days after the first vaccination. ^(o)Standardchemotherapy will be given only if needed for progressive disease. Theregimen will be at the discretion of the investigator. ^(p)All adverseevents and grade ≧3 laboratory toxicities will be recorded at eachvisit. ^(q)Fludarabine 30 mg/m² IV daily over 45 minutes for 3 daysfollowed by fTBI. ^(r)The dose of the fTBI will be determined accordingto the cohort the patient enters into and will be given once daily for2-4 days ^(s)Autologous hematopoietic and immune cell rescue (HICR):thawed cells will be transfused as rapidly as possible. Patients will bepre-medicated with diphenhydramine 50 mg IV and hydrocortisone 100 mg IV30 minutes before the transfusion. ^(t)Acyclovir prophylaxis: if HSV-1or -2 positive, Acyclovir 400 mg p.o. bid starting on the first day offTBI ^(u)Bacterial prophylaxis: When ANC < 500, start Ciprofloxacin 500mg orally daily. Days +30-60: Bactrim 160 mg/800 mg p.o. bid, Saturdayand Sunday only for pneumocystis carinii prophylaxis. ^(v)Serum CMVmonitoring once weekly for 1 year ^(w)Immune monitoring: See separateschedule in section 4.1.16.3. Immune monitoring will be done at month+1, +3, +6, +12, +18, and at termination of the study. ^(x)Routinelaboratory monitoring per institution's standard practice for safety oras mandated by patients co-morbidities. ^(y)After study termination,patients will be followed for survival data approximately every 3months. When possible, subsequent treatment history will be recorded.Measurement of Effect

Anti-Tumor Effect

Patients will undergo re-staging CT scans of the chest/abdomen/pelvis atweek 4 and week 8 to decide if standard chemotherapy is needed. Ifchemotherapy is initiated or continued at week 8, then the patient willthen have a repeat CT scan of the chest/abdomen/pelvis after completionof at least 3 cycles of standard chemotherapy. After transplant,patients will undergo monthly CT scans of the chest/abdomen/pelvis for 2months then every 2 months and as needed per the Investigators'discretion to assess for progressive disease or response. Thepost-transplant month 2 CT scan will be considered the confirmatoryresponse scan.

Patients will be evaluable for toxicity from day 1 of baselineapheresis.

Patients will be evaluable for objective response by CT imaging atseveral time points:

1. Week 8 if not on chemotherapy.

2. One, 2, and 4 months after autologous hematopoietic and immune cellrescue.

The post-rescue CT scans will be compared to both the pre-conditioningand screening CT scans. Responses from the pre-conditioning scans (week8 or 18) as compared to the post-rescue scans will be the primaryassessment of response to our investigational therapy as the comparisonto the study entry (screening) scan may be biased by any chemotherapyintervention.

Efficacy assessment will be done using Response Evaluation Criteria inSolid Tumors (RECIST) (92).

Disease Parameters

Accurate estimation of the overall tumor burden at baseline is necessaryto assess objective response with treatment. Measurable disease isdefined by the presence of at least one measurable lesion.

All measurements will be recorded in metric notation by use of ruler,calipers or computer-assisted measurement tools. The same method ofassessment and the same technique should be used to characterize eachidentified lesion at baseline and during follow-up. All baselineevaluations should be performed no more than four weeks beforeregistration.

Definition of Measurable Disease

Lesions that can be accurately measured in at least one dimension(longest dimension to be recorded) as ≧20 mm (2.0 cm) with conventionaltechniques or as ≧10 mm (1.0 cm) with a spiral CT scan.

For measurable disease existing as a solitary lesion, confirmation ofmalignant nature should be performed with cytology/histology if there isreasonable doubt in the investigator's opinion about the origin of thelesion.

Definition of Non-Measurable Disease

All lesions not considered measurable by the definition above, includingsmall lesions (longest diameter ≦20 mm with conventional techniques or≦10 mm with spiral CT) and truly non-measurable lesions. Trulynon-measurable lesions include: bone lesions, leptomeningeal disease,malignant ascites, malignant pleural or pericardial effusion,inflammatory breast disease, lymphangitic spread of tumor, and abdominalmasses that are not pathologically confirmed metastases and followedsolely by imaging techniques.

Previously irradiated lesions are considered non-measurable.

Methods for Evaluation of Measurable Disease

CT and MRI: CT and MRI scans are currently the best available and mostreproducible methods for measuring target lesions. Conventional CT andMRI should be performed with contiguous cuts of 10 mm or less in slicethickness. Spiral CT should be performed using a 5 mm contiguousreconstruction algorithm.

Chest X-ray: lesions on chest X-ray are acceptable as measurable lesionswhen they are clearly defined and surrounded by aerated lung.

Clinical examination: clinically detected lesions will only beconsidered measurable if they are superficial and readily palpable onrepeated clinical examination.

Response Criteria

Evaluation of Target Lesions

Target lesions will be defined as all measurable lesions up to a maximumof five lesions per organ and 10 lesions in total, representative of allinvolved organs. Target lesions should be selected based on the largestsize and best suitability for accurate repeated measurements.

The sum of the longest diameters of all target lesions will becalculated at baseline and reported as the baseline sum longestdiameter. This baseline sum longestdiameter will be used as thereference to characterize objective tumor response. For lesionsmeasurable in 2 or 3 dimensions, the longest diameter at the time ofassessment will be reported.

Using RECIST criteria, target lesions will be evaluated:

-   -   Complete response (CR): The disappearance of all target and        non-target lesions, and no new lesions. If any tumor markers        were elevated prior to therapy they must be normal for the        patient to be declared a CR.    -   Partial response (PR): At least a 30% decrease in the sum of the        longest diameters of target lesions, taking as reference the        baseline sum longest diameter.    -   Progressive disease (PD): At least a 20% increase in the sum of        the longest diameters of the target lesions, taking as reference        the smallest sum longest diameter recorded since the baseline        measurements, or the appearance of one or more new lesions.    -   Stable disease (SD): Neither sufficient shrinkage to qualify for        partial response nor sufficient increase to qualify for        progressive disease. To be assigned a status of stable disease,        measurements must have met the stable disease criteria at least        once after study entry at a minimum interval of six weeks.    -   Symptomatic deterioration: Patients with global deterioration of        health status requiring discontinuation of treatment without        objective evidence of disease progression at that time should be        classified as having symptomatic deterioration.

Evaluation of Best Overall Response

The best overall response is the best response recorded fromregistration until disease progression/recurrence.

Target Lesions Nontarget Lesions New Lesions Overall Response CR CR NoCR CR PR/SD No PR PR No PD No PR SD No PD No SD PD Any Yes or No PD AnyPD Yes or No PD Any Any Yes PD CR = complete response; PR = partialresponse; SD = stable disease; PD = progressive disease

To be assigned a status of stable disease, measurements must have metthe stable disease criteria at least once after study entry at a minimuminterval of six weeks.

Duration of Response

Duration of overall response is the period measured from the time thatmeasurement criteria are met for complete or partial response (whicheverstatus is recorded first) until the first date that recurrent orprogressive disease is objectively documented, taking as reference thesmallest measurements recorded since treatment started.

Other Efficacy Parameters

First documentation of Response: The time between initiation of therapyand first documentation of PR or CR.

Duration of Stable Disease: Duration of stable disease is themeasurement from registration until the criteria for disease progressionis met, taking as reference the smallest measurements recorded sinceregistration. To be assigned a status of stable disease, measurementsmust have met the stable disease criteria at least once after studyentry at a minimum interval of six weeks.

-   -   Overall Survival (OS): Time from the date of enrollment to the        date of death due to any cause or the last date the patient was        known to be alive (censored observation) at the date of data        cutoff for the final analysis    -   Time to Progression (TTP): Time from the date of enrollment to        the date of the first observation of documented disease        progression or death to due cancer        Other Response Parameters

Tumor markers: Tumor markers alone cannot be used to assess response.However, if a tumor marker is initially above the upper limit of normal,it must normalize for a patient to be considered in complete CR when alltumor lesions have disappeared. The tumor markers that will be assessedin this study are CEA and CA 19-9.

Statistical Considerations

Endpoints

Primary Endpoint

To assess the feasibility of using an autologous tumor cell vaccine incombination with standard chemotherapy and investigational autologoushematopoietic and immune cell rescue in terms of acceptable clinicaltoxicity.

Secondary Endpoints

-   -   Ex vivo assessment of immune response    -   Response    -   TTP        Plan of Analysis

Background and Demographic Characteristics

Baseline demographic characteristics will be recorded on each patientincluding age, gender, race, ECOG performance status, location ofprimary cancer, location and number of metastases, presence or absenceof primary tumor, dates of initial diagnosis and recurrence ifapplicable, and presence of other co-morbidities.

Evaluation of Safety

Any patient who receives the baseline apheresis will be included in thequalitative safety analysis irrespective of whether they receivevaccination.

Evaluation of Efficacy

The study will not be powered for efficacy but we will determineresponse according to RECIST criteria and PFS to qualitatively assessfor therapeutic promise.

Methods for Handling Missing Data and Non-Adherence to Protocol

If missing data is discovered, genuine efforts will be taken to recoverthe data when possible. If protocol violations occur, they will bereported to the IRB, SRC, FDA, and Pfizer.

Interim Analyses

Weekly meetings of the investigative team will be held to review thepatients on trial and to discuss all toxicities encountered. Safety datawill be reviewed continuously. If the trial is not deemed feasible froma safety or financial standpoint or is not thought efficacious, it willbe halted prior to complete accrual of the 10 patients if not alreadydone.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

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What is claimed is:
 1. A method for treating cancer of a subjectcomprising: (a) obtaining purified solid tumor cells from the subjectwherein the solid tumor cells are selected from the group consisting ofcolorectal tumor, lung tumor, breast tumor, pancreatic tumor, livertumor, prostate tumor, and ovarian tumor; (b) vaccinating the subjectwith cancer with said purified solid tumor cells that have beenirradiated and formulated in combination with an adjuvant; (c)collecting immune and hematopoietic cells from the vaccinated subject;(d) irradiating said subject to myeloablative total body irradiation;and (e) injecting the collected immune and hematopoietic cells from thesubject intravenously into said subject, without ex vivo T cellexpansion or treatment with cytokines.
 2. The method of claim 1 whereinthe tumor cells are purified away from stromal cells.
 3. The method ofclaim 1 wherein the tumor cells are purified away from immunosuppressivecells.
 4. The method of claim 1 wherein the purified tumor cells arestimulated with adjuvant prior to the vaccinating.
 5. The method ofclaim 1, wherein the adjuvant is CpG or GM-CSF.
 6. The method of claim 1wherein the immune and hematopoietic cells collected from the vaccinatedsubject comprise T cells.
 7. The method of claim 1 wherein the immunecells comprise hematopoietic progenitor cells.
 8. The method of claim 7wherein the progenitor cells comprise CD34+ cells.
 9. The method ofclaim 1 wherein the hematopoietic cells are mobilized in the vaccinatedsubject and enriched prior to the step of injecting the collected immuneand hematopoietic cells.
 10. The method of claim 1 wherein the recipientreceives said total body irradiation with local irradiation of thetumor.
 11. The method of claim 1 wherein the cancer is colorectalcancer.
 12. The method of claim 1 wherein the tumor cells are from aprimary or metastatic tumor.